U.S. patent application number 14/266540 was filed with the patent office on 2014-10-23 for antibody producing non-human mammals.
The applicant listed for this patent is MERUS B.V.. Invention is credited to Cornelis A. DE KRUIF, Erwin HOUTZAGER, Robert A. KRAMER, Ton LOGTENBERG, Rui Daniel PINTO, Mark THROSBY.
Application Number | 20140314755 14/266540 |
Document ID | / |
Family ID | 42007787 |
Filed Date | 2014-10-23 |
United States Patent
Application |
20140314755 |
Kind Code |
A1 |
LOGTENBERG; Ton ; et
al. |
October 23, 2014 |
ANTIBODY PRODUCING NON-HUMAN MAMMALS
Abstract
Described are transgenic, non-human animals comprising a nucleic
acid encoding an immunoglobulin light chain, whereby the
immunoglobulin light chain is human, human-like, or humanized. The
nucleic acid is provided with a means that renders it resistant to
DNA rearrangements and/or somatic hypermutations. In one
embodiment, the nucleic acid comprises an expression cassette for
the expression of a desired molecule in cells during a certain
stage of development in cells developing into mature B cells.
Further provided is methods for producing an immunoglobulin from
the transgenic, non-human animal.
Inventors: |
LOGTENBERG; Ton; (Utrecht,
NL) ; THROSBY; Mark; (Utrecht, NL) ; KRAMER;
Robert A.; (Utrecht, NL) ; PINTO; Rui Daniel;
(Utrecht, NL) ; DE KRUIF; Cornelis A.; (Utrecht,
NL) ; HOUTZAGER; Erwin; (Zeist, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MERUS B.V. |
Utrecht |
|
NL |
|
|
Family ID: |
42007787 |
Appl. No.: |
14/266540 |
Filed: |
April 30, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12589181 |
Oct 19, 2009 |
|
|
|
14266540 |
|
|
|
|
12459285 |
Jun 29, 2009 |
|
|
|
12589181 |
|
|
|
|
61133274 |
Jun 27, 2008 |
|
|
|
Current U.S.
Class: |
424/133.1 |
Current CPC
Class: |
A01K 2217/206 20130101;
A01K 2267/01 20130101; A01K 2207/15 20130101; A01K 2227/105
20130101; C12P 21/00 20130101; A01K 2217/075 20130101; C07K 14/47
20130101; C07K 16/248 20130101; A01K 2217/15 20130101; C12N 15/8509
20130101; A01K 67/0278 20130101; A01K 67/027 20130101; A01K 67/0275
20130101; C07K 16/1282 20130101; C07K 2317/24 20130101; A01K
2217/052 20130101; C07K 16/462 20130101 |
Class at
Publication: |
424/133.1 |
International
Class: |
C07K 16/24 20060101
C07K016/24; C07K 16/12 20060101 C07K016/12 |
Claims
1-30. (canceled)
31. A method of obtaining an antibody that binds to an antigen, the
method comprising immunizing a transgenic mouse with the antigen,
wherein the genome of the transgenic mouse comprises a human
immunoglobulin light chain germline V gene segment joined to a
human immunoglobulin light chain germline J gene segment linked to
a murine light chain constant gene region, wherein the joined human
V/J gene segments encode a rearranged human immunoglobulin light
chain variable region; identifying a rearranged immunoglobulin
heavy chain variable region produced by the transgenic mouse that,
when paired with the rearranged human immunoglobulin light chain
variable region, specifically binds to the antigen; expressing
nucleic acids encoding the rearranged immunoglobulin heavy chain
variable region and the rearranged human immunoglobulin light chain
variable region in a host cell; and obtaining the antibody.
32. The method of claim 31, wherein the human immunoglobulin light
chain V gene segment is a human V.kappa. germline gene segment, the
J gene segment is a human J.kappa. gene segment, and the murine
constant region is a .kappa. light chain constant region.
33. The method of claim 32, wherein the human germline V.kappa.
gene segment is IGV.kappa.1-39.
34. The method of claim 32, wherein the human germline J.kappa.
gene segment is IGJ.kappa.1.
35. The method of claim 31, wherein the rearranged immunoglobulin
heavy chain variable region is a human rearranged immunoglobulin
heavy chain variable region.
36. The method of claim 31, wherein at least one endogenous
immunoglobulin light chain locus of the transgenic mouse is
functionally silenced.
37. The method of claim 32, wherein the endogenous .kappa. light
chain locus of the transgenic mouse is functionally silenced.
38. The method of claim 31, further comprising isolating a B cell
expressing the rearranged immunoglobulin heavy chain variable
region from the immunized transgenic mouse.
39. The method of claim 38, further comprising immortalizing the
isolated B cell to produce a hybridoma.
40. The method of claim 38, further comprising obtaining a nucleic
acid encoding the rearranged immunoglobulin heavy chain variable
region from the isolated B cell.
41. The method of claim 31, wherein the rearranged human light
chain variable region comprises the amino acid sequence of the
Vk1-39 region of SEQ ID NO: 85.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of U.S. patent application
Ser. No. 12/589,181, filed Oct. 19, 2009, pending, which is a
continuation of U.S. patent application Ser. No. 12/459,285, filed
Jun. 29, 2009, abandoned, which claims the benefit, under 35 U.S.C.
.sctn.119(e), to U.S. Provisional Patent Application Ser. No.
61/133,274, filed Jun. 27, 2008, for "Antibody Producing Non-Human
Mammals," the entire contents of each of which are hereby
incorporated herein by this reference.
STATEMENT ACCORDING TO 37 C.F.R. .sctn.1.821(C) OR (E)--SEQUENCE
LISTING SUBMITTED AS ASCII TEXT FILE
[0002] Pursuant to 37 C.F.R. .sctn.1.821(c) or (e), files
containing a TXT version and a PDF version of the Sequence Listing
have been submitted concomitant with this application, the contents
of which are hereby incorporated by reference.
TECHNICAL FIELD
[0003] The invention relates to the production and use of non-human
animals capable of producing antibodies or derivatives thereof,
which are expressed from at least partially exogenous nucleic acids
(transgenes). Transgenes to produce such transgenic animals and
methods to produce such heterologous antibodies; methods and
vectors for producing such transgenic animals are disclosed.
BACKGROUND
[0004] B cells mediate humoral immunity by producing specific
antibodies. The basic structural subunit of an antibody (Ab) is an
immunoglobulin (Ig) molecule. Ig molecules consist of a complex of
two identical heavy (H) and two identical light (L) polypeptide
chains. At the amino terminus of each H chain and L chain is a
region that varies in amino acid sequence named the variable (V)
region. The remaining portion of the H and L chains is relatively
constant in amino acid sequence and is named the constant (C)
region. In an Ig molecule, the H and L chain V regions (VH and VL)
are juxtaposed to form the potential antigen-binding site. The
genes that encode H and L chain V regions are assembled somatically
from segments of germline DNA during precursor B (pre-B) cell
differentiation: V, D and J gene segments for the H chain and V and
J gene segments for the L chain. Within Ig V regions are three
regions of greatest amino acid sequence variability that interact
to form the antigen-recognition site and are thus referred to as
complementarity determining regions (CDRs).
[0005] The V gene segment encodes the bulk of the V region domain,
including CDR1 and CDR2. Diversity in CDR1 and CDR2 derives from
sequence heterogeneity among multiple different germline-encoded V
segments. CDR3 is encoded by sequences that are formed by the
joining of H chain V, D, and J gene segments and L chain V and J
segments and by mechanisms that create nucleotide sequence
heterogeneity where these segments are combined. Additional
diversity may be derived from pairing of different H and L chain V
regions. Collectively these processes yield a primary repertoire of
antibodies encoded by germline gene segments and expressed by newly
formed B cells.
[0006] An additional source of antibody diversity is imposed on top
of the diversity generated by recombination of Ig gene segments. B
cells are able to introduce mutations into the antibody V regions
that they express, a process called somatic hypermutation. Thus,
when an animal first encounters an antigen, the antigen binds to a
specific B cell which happens to carry antibodies which have a V
domain which binds the antigen. This primary response may activate
this B cell to go on to secrete the cognate antibody. These
activated B cells can also now target a somatic mutation process to
their rearranged antibody gene segments and thus allow the
production of daughter cells which make variants of the antibodies
of the primary response. A selection process amplifies those
variant B cell descendants which make an antibody of improved
affinity of the antigen. In B cells, somatic hypermutations are
targeted to a restricted genomic region including both the
rearranged VH and VL genes. Thus somatic mutation allows affinity
maturation--the production and selection of high affinity
antibodies. Therefore, somatic mutation is important for the
generation of high affinity antibodies.
[0007] The exquisite specificity and high affinity of antibodies
and the discovery of hybridoma technology allowing the generation
of monoclonal antibodies (mAbs) has generated great expectations
for their utilization as targeted therapeutics for human diseases.
MAbs are identical because they are produced by a single B cell and
its progeny. MAbs are made by fusing the spleen cells from a mouse
that has been immunized with the desired antigen with myeloma cells
to generate immortalized hybridomas. One of the major impediments
facing the development of in vivo applications for mAbs in humans
is the intrinsic immunogenicity of non-human Igs. Patients respond
to therapeutic doses of mouse mAbs by making antibodies against the
mouse Ig sequences (Human Anti Mouse Antibodies; HAMA), causing
acute toxicity, alter their biodistribution and accelerate
clearance, thus reducing the efficacy of subsequent administrations
(Mirick et al. (2004), Q. Nucl. Med. Mol. Imaging 48:251-257).
[0008] To circumvent the generation of HAMA, antibody humanization
methods have been developed in an attempt to produce mAbs with
decreased immunogenicity when applied to humans. These endeavors
have yielded various recombinant DNA-based approaches aimed at
increasing the content of human amino acid sequences in mAbs while
retaining the specificity and affinity of the parental non-human
antibody. Humanization began with the construction of mouse-human
chimeric mAbs (S. L. Morrison et al. (1984), Proc. Natl. Acad. Sci.
USA 81:6851-5), in which the Ig C regions in murine mAbs were
replaced by human C regions. Chimeric mAbs contain 60-70% of human
amino acid sequences and are considerably less immunogenic than
their murine counterparts when injected into humans, albeit that a
human anti-chimeric antibody response was still observed (W. Y.
Hwang et al. (2005), Methods 36:3-10).
[0009] In attempts to further humanize murine mAbs, CDR grafting
was developed. In CDR grafting, murine antibodies are humanized by
grafting their CDRs onto the VL and VH frameworks of human Ig
molecules, while retaining those murine framework residues deemed
essential for specificity and affinity (P. T. Jones et al. (1986),
Nature 321:522). Overall, CDR-grafted antibodies consist of more
than 80% human amino acid sequences (C. Queen et al. (1989), Proc.
Natl. Acad. Sci. U.S.A. 86:10029; P. Carter et al. (1992), Proc.
Natl. Acad. Sci. U.S.A. 89:4285). Despite these efforts,
CDR-grafted, humanized antibodies were shown to still evoke an
antibody response against the grafted V region (W. Y. Hwang et al.
(2005), Methods 36:3).
[0010] Subsequently to CDR grafting, humanization methods based on
different paradigms such as resurfacing (E. A. Padlan et al.
(1991), Mol. Immunol. 28:489), superhumanization (P. Tan D. A. et
al. (2002), J. Immunol. 169:1119), human string content
optimization (G. A. Lazar et al. (2007), Mol. Immunol. 44:1986) and
humaneering have been developed in an attempt to further decrease
the content of non-human sequences in therapeutic mAbs (J. C.
Almagro et al. (2008), Frontiers in Bioscience 13:1619). As in CDR
grafting approaches, these methods rely on analyses of the antibody
structure and sequence comparison of the non-human and human mAbs
in order to evaluate the impact of the humanization process into
immunogenicity of the final product. When comparing the
immunogenicity of chimeric and humanized antibodies, humanization
of variable regions appears to decrease immunogenicity further (W.
Y. Hwang et al. (2005), Methods 36:3-10).
[0011] De-immunization is another approach developed to reduce the
immunogenicity of chimeric or mouse antibodies. It involves the
identification of linear T-cell epitopes in the antibody of
interest, using bioinformatics, and their subsequent replacement by
site-directed mutagenesis to human or non-immunogenic sequences (WO
9852976 A1, the contents of which are incorporated by this
reference). Although de-immunized antibodies exhibited reduced
immunogenicity in primates, compared with their chimeric
counterparts, some loss of binding affinity was observed (M. Jain
et al. (2007), Trends in Biotechnol. 25:307).
[0012] The development of phage display technology complemented and
extended humanization approaches in attempts to obtain less
immunogenic mAbs for therapy in humans. In phage display, large
collections ("libraries") of human antibody VH and VL regions are
expressed on the surface of filamentous bacteriophage particles.
From these libraries, rare phages are selected through binding
interaction with antigen; soluble antibody fragments are expressed
from infected bacteria and the affinity of binding of selected
antibodies is improved by mutation (G. Winter et al. (1994), Annu.
Rev. Immunol. 12:433). The process mimics immune selection, and
antibodies with many different bindings specificities have been
isolated using this approach (H. R. Hoogenboom et al. (2005), Nat.
Biotechnol. 23:1105). Various sources of H and L chain V regions
have been used to construct phage display libraries including those
isolated from non-immune or immune donors. In addition, phage
display libraries have been constructed of V regions that contain
artificially randomized synthetic CDR regions in order to create
additional diversity. Often, antibodies obtained from phage display
libraries are subjected to in vitro affinity maturation to obtain
high affinity antibodies (H. R. Hoogenboom et al. (2005), Nat.
Biotechnol. 23:1105).
[0013] The creation of transgenic mouse strains producing human
antibodies in the absence of mouse antibodies has provided another
technology platform for the generation of specific and high
affinity human mAbs for application in humans. In these transgenic
animals, the endogenous mouse antibody machinery is inactivated and
replaced by human Ig loci to substantially reproduce the human
humoral immune system in mice (A. Jakobovits et al. (2007), Nat.
Biotechnol. 25:1134; N. Lonberg (2005), Nat. Biotechnol. 23:1117).
B cell development as well as Ig diversification by recombination
of gene segments is faithfully reproduced in these mice, leading to
a diverse repertoire of murine B cells expressing human Igs. By
immunizing these mice with antigens, it was further demonstrated
that these transgenic animals accumulated somatic mutations in the
V regions of both heavy and light chains to produce a wide
diversity of high-affinity human mAbs (N. Lonberg (2005), Nat.
Biotechnol. 23:1117).
[0014] The question, whether "fully human" mAbs such as derived
from phage display libraries or transgenic mice are less
immunogenic than humanized mAbs cannot be answered yet, because
full immunogenicity data are available for just two human mAbs. An
anti-tumor necrosis factor mAb, developed from phage-displayed
human libraries induced antibody responses in 12% of patients--at
the higher end of the incidence of anti-antibody responses of the
humanized antibodies (W. Y. Hwang et al. (2005), Methods
36:3-10).
[0015] Evaluation of the immunogenicity of the first registered
human mAb generated by the transgenic approach demonstrated that
mAb treatment resulted in the generation of antibodies in
approximately 5.5% of treated cancer patients (A. Jakobovits et al.
(2007), Nat. Biotechnol. 25:1134; J. A. Lofgren et al. (2007), J.
Immunol. 178:7467).
DISCLOSURE OF THE INVENTION
[0016] Disclosed are a method and means for producing antibodies
that are specific for their targets, but are less immunogenic.
Described herein, the reduction of immunogenicity is at least
partially achieved by providing a transgenic non-human mammal
comprising, at least in its B cell lineage, a nucleic acid encoding
at least an immunoglobulin light chain or heavy chain, wherein the
heavy- or light chain encoding sequence is provided with a means
that renders it resistant to DNA rearrangements and/or somatic
hypermutations, preferably such a non-human animal is a rodent,
more specifically a mouse. In certain embodiments, the nucleic acid
encodes a human, human-like, or humanized immunoglobulin chain.
[0017] In the remainder of this specification, mice are typically
used as examples of the non-human mammals. The transgenic,
non-human, mammalian hosts are capable of mounting an immune
response to an antigen, where the response produces antibodies
having primate, particularly human, variable regions. Various
transgenic hosts may be employed, particularly murine, lagomorpha,
ovine, avine, porcine, equine, canine, feline, or the like. Mice
have been used for the production of B-lymphocytes for
immortalization for the production of antibodies. Since mice are
easy to handle, can be bred in large numbers, and are known to have
an extensive immune repertoire, mice will usually be the animal of
choice. Therefore, in the following discussion, the discussion will
refer to mice, but it should be understood that other animals,
particularly non-primate mammals, may be readily substituted for
the mice, following the same procedures.
[0018] The reason for preventing rearrangements and hypermutation
is that in this manner a non-immunogenic polypeptide can be chosen
beforehand knowing that this polypeptide chain will remain
non-immunogenic. At least one of the chains of the resulting
immunoglobulin is thus less immunogenic. The resulting antibody
needs to have (usually) both a light- and a heavy chain. The
non-immunogenic chain must therefore be capable of pairing with the
other chain. The other chain may be an endogenous chain, an
exogenous chain or a hybrid of both. For human therapy, the
non-immunogenic chain should be as close to human as possible.
[0019] A means for rendering a gene encoding an immunoglobulin
chain (or chains) resistant to DNA rearrangement and/or mutation is
of course removal of all genetic elements responsible for the
rearrangement and/or mutation. The drawback thereof is that the
variability of the two chains is eliminated, whereas the invention
preferably retains the variability in one chain (preferably the
heavy chain) and inhibits and/or prevents the
rearrangement-mutation of the other chain (preferably the light
chain).
[0020] The elements for rearrangement and/or hypermutation
characterized so far are located within the loci for
immunoglobulins. Therefore the means for rendering the
immunoglobulin encoding sequence resistant to DNA rearrangement
and/or mutation is inserting the gene in a locus outside the
immunoglobulin loci.
[0021] Thus, described herein, a transgenic non-human mammal is
provided wherein the light/heavy chain encoding sequence is
integrated in the genome of the non-human mammal in a locus outside
the immunoglobulin loci. Preferably the insertion is in a locus
that is resistant to gene silencing. Described herein, the
integration is in the Rosa-locus or a comparable locus.
[0022] In certain embodiments, provided is an expression cassette
that can be inserted into a Rosa locus or comparable locus with a
means that allows expression of the immunoglobulin chain(s)
essentially limited to cells of B cell lineage, preferably with a
means that allows expression of the light chain encoding nucleic
acid during a certain stage of the development of B cells. The term
"essentially limited expression" indicates that expression is
predominantly in cells of the B-cell lineage, but that lower levels
of expression in other cells, as compared to the level of
expression in B-cells, is possible. In certain embodiments, the
term "essentially limited expression" indicates that the expression
is exclusively present in cells of the B-cell lineage. Such means
typically and preferably include B cell (developmental stage)
specific promoters such as CD19, CD20, .mu.HC (all V-genes),
VpreB1, VpreB2, VpreB3, .lamda.5, Ig.alpha., Ig.beta., .kappa.LC
(all genes), .lamda.LC (all genes), BSAP (Pax5). Although it is
very well possible to direct the expression of the DNA
rearrangement and/or mutation resistant chain by such promoters,
they are relatively weak. A strong promoter will typically be
required to ensure adequate surface expression of the B cell
receptor (made up of the membrane attached Ig H and L chain) and to
compete with the expression and pairing of endogenous chains (if
present) through allelic exclusion. Such a promoter, however is
usually not tissue specific. To confer tissue specificity, an
indirect system employing Cre/lox or the like is preferred. The
desired chain is put under control of a strong promoter inhibited
by an element that can be removed by the action of a Cre-protein,
leading to activation of the desired immunoglobulin encoding gene.
This system is described in detail in F. T. Wunderlich (2004),
"Generation of inducible Cre systems for conditional gene
inactivation in mice," Inauguraldissertation zur Erlangung des
Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultat der
Universitat zu Koln; on the internet at
deposit.ddb.de/cgi-bin/dokserv?idn=97557230x&dok_var=d1&dok_ext=pdf&filen-
ame=975572 30x.pdf.
[0023] Preferably the immunoglobulin chain produced in a manner
resistant to rearrangements and hypermutation is a light chain
capable of pairing with different heavy chains encoded by the
non-human mammal. The light chain will then be the same (and less
immunogenic) in all antibodies, but variety in specificity is
retained through rearrangements and hypermutations in the heavy
chains. It may in that case be preferable to silence at least one
of the endogenous loci encoding a light chain, although allelic
exclusion may render this unnecessary.
[0024] According to this embodiment, preferably the endogenous
kappa (.kappa.) light chain locus is functionally silenced.
[0025] If the endogenous .kappa. light chain locus is silenced, but
also for other reasons, it is preferred that the resistant light
chain is a .kappa. light chain, preferably a light chain that has a
germline-like sequence. Described herein such a light chain would
lead to an antibody with reduced immunogenicity. The preferred
germline sequence is based on the human IGKV1-39 (O12) as this
light chain is very frequently observed in the human repertoire (de
Wildt et al. 1999, J. Mol. Biol. 285(3):895 and has superior
thermodynamic stability, yield and solubility (Ewert et al. 2003,
J. Mol. Biol. 325(3):531).
[0026] The following gives more specific embodiments of the
expression cassette with which the non-human animal can be provided
described herein. Although this is typically advantageous for
immunoglobulins, other genes of interest are also contemplated.
[0027] Thus, provided in a specific embodiment, is a transgenic
non-human mammal wherein the light chain encoding nucleic acid
comprises in 5'-3' direction: a B cell specific promoter, a leader,
a rearranged human V gene, optionally a mouse .kappa.-intron
enhancer (MoE.kappa.i), a constant region (.kappa.) and optionally
a (truncated) mouse .kappa.-3' enhancer (MoE.kappa.3'). Neuberger
identified and examined a novel B-cell specific enhancer located
downstream of the kappa constant region (Neuberger, EP 00469025 B1,
the contents of which are incorporated herein by this reference).
This enhancer has been shown to play a crucial role in the
expression of kappa genes as removal of the 808 bp enhancer
strongly reduced expression. Deletion of the 3' kappa enhancer also
strongly reduced the level of somatic hypermutations (SHM). In
transgenic and cell expression studies, it has been revealed that
reduced, mutated or deleted 3' kappa enhancers not only lowered
expression levels, but also decreased the level of somatic
hypermutations. Currently, it cannot be determined whether the 3'
kappa enhancer is involved in SHM processes, expression regulation
or both (review V. H. Odegard et al. (2006), Nat. Rev. Immunol.
6:573; M. Inlay et al. (2002), Nat. Immunol. 3:463).
[0028] Detailed expression studies using engineered variants of the
3' kappa enhancer indicated that a 50 nucleotide region is
sufficient to drive expression. However for proper expression a
reduced sequence of 145 nucleotides is preferred (EP04690251; K. B.
Meyer et al. (1990), Nucleic Acids Res. 18(19):5609-15).
[0029] Thus, the invention in one aspect provides a nucleic acid
for insertion into the genome of a non human animal that is an
expression cassette for the expression of a desired proteinaceous
molecule in cells developing into mature B cells during a certain
stage of development, the cassette comprising means for preventing
silencing of expression of the desired proteinaceous molecule after
introduction into a host cell, and means for timing expression of
the desired proteinaceous molecule with the desired developmental
stage of the host cell.
[0030] An expression cassette is defined as a nucleic acid that has
been provided with means for introduction into the genome of a host
cell, such as sequences which allow for homologous recombination
with a certain site in the genome. Usually the nucleic acid will be
DNA, typically double stranded. Typically the expression cassette
will be provided to the cell in a vector from which it is
transferred to the genome of the cell. The expression cassette
further comprises all elements necessary for expression of the gene
in a host cell, although in certain embodiments some of such
elements may be present on a second nucleic acid to be introduced,
whereby these elements act in trans. Elements necessary for
expression in a host cell include promoters, enhancers and other
regulatory elements. Only those elements are necessary that are not
provided by the host cell.
[0031] The expression of the gene of interest should not be
silenced in the genome of the host cell, especially not in the
development stage where expression is required. This can be done by
various means, such as insertion into the endogenous locus or by
providing the cassette with nucleic acid elements that prevent
silencing (Kwaks et al. (2006), Trends Biotechnol. 24(3):137-142,
which is incorporated herein by reference). It is preferred that
the expression cassette is inserted in a locus that is not silenced
in the host cells (EP 01439234; which is incorporated herein by
reference).
[0032] The means for prevention of silencing comprise STabilizing
Anti-Repression-sequences (STAR.RTM.-sequences) and Matrix
Attachment Regions (MARs). A STAR sequence is a nucleic acid
sequence that comprises a capacity to influence transcription of
genes in cis. Typically, although not necessarily, a STAR sequence
does not code by itself for a functional protein element. In one
embodiment one STAR element is used. Preferably, however, more than
one STAR element is used. In a particularly preferred embodiment an
expression cassette described herein is provided with two STAR
sequences; one STAR sequence at the 5' side of the coding sequence
of the immunoglobulin gene and one STAR sequence at the 3' side of
the coding sequence of the immunoglobulin gene. MARs are DNA
sequences that are involved in anchoring DNA/chromatin to the
nuclear matrix and they have been described in both mammalian and
plant species. MARs possess a number of features that facilitate
the opening and maintenance of euchromatin. MARs can increase
transgene expression and limit position-effects.
[0033] Expression from the cassette should only occur during a
certain period in the development of a cell, in particular a
developing B cell, more in particular a B cell in a transgenic
non-human animal, in particular a mouse. In this particular case
the developmental period is chosen such that the expression of the
gene from the cassette (typically a light- or heavy chain-like
polypeptide) does not significantly interfere with the normal
differentiation and/or maturation of the cell and when applicable,
allows for pairing of the polypeptide chain produced with its
counterpart.
[0034] This may, in one embodiment, be achieved by providing a
nucleic acid described herein, wherein the means for timing
expression is a promoter of which the activity is essentially
limited to the certain stage of development. In a developing B
cell, which, e.g., after immunization is maturing and/or
differentiating, the expression of the gene of interest, when it is
one of the polypeptide chains of an immunoglobulin, must not
interfere (significantly) with the maturation and/or
differentiation and it needs to be timed such that the resulting
polypeptide can pair with its counterparts. Therefore, provided is
a nucleic acid described herein wherein the certain stage starts at
a stage immediately preceding or coinciding with the onset of the
expression of light chain molecules by the cells at a certain stage
of development into a mature B cell. This may be achieved by
selecting a promoter which is active only during the suitable
period. Such a promoter may be a CD19 promoter, the Ig-.alpha.
promoter, the Ig-.beta. promoter, the .mu.hc (all genes) promoter,
the Vk promoter or analogues or homologues thereof.
[0035] In a specific embodiment, the promoter as disclosed above
does not drive the expression of the gene of interest directly.
Instead it drives the expression of a gene of which the product
activates in trans the expression of the gene of interest. Such an
activating gene may be a gene encoding a so-called Cre recombinase
or Cre-like protein. The expression cassette for the gene of
interest may, e.g., be provided with a sequence that inhibits
expression of the gene of interest. The sequence can be removed by
the action of the Cre recombinase, which is under control of the
desired promoter (active during the proper stage of development).
In this embodiment a set of expression cassettes is required.
[0036] Therefore, provided is a set of nucleic acids that are
expression cassettes, wherein one nucleic acid comprises an
expression cassette encoding a Cre-like protein under control of a
promoter active during the desired stage of development of the host
cell and the second nucleic acid comprises a sequence encoding a
desired proteinaceous molecule under control of a constitutive
promoter which can be activated by the action of a Cre-like
protein. The activation is preferably achieved by removal of a stop
sequence flanked by loxP sites. The Cre/lox system is described in
detail in Rajewsky et al. (1996), J. Clin. Invest. 98:600-603,
which is incorporated herein by reference. Such systems are
reviewed in F. T. Wunderlich (2004), "Generation of inducible Cre
systems for conditional gene inactivation in mice,"
Inauguraldissertation zur Erlangung des Doktorgrades der
Mathematisch-Naturwissenschaftlichen Fakultat der Universitat zu
Koln; on the World Wide Web at
deposit.ddb.de/cgi-bin/dokserv?idn=97557230x&dok_var=d1&dok_ext=pdf&filen-
ame=97557230x.pd, which is incorporated herein by reference.
[0037] Further provided is a transgenic non-human animal that has
been provided with an expression cassette hereof, wherein the
desired proteinaceous molecule is a polypeptide chain of an
immunoglobulin. A preferred polypeptide chain is a light chain. A
more preferred polypeptide is a germline or germline-like light
chain. A most preferred polypeptide is encoded by the
immunoglobulin kappa variable 1-39 (IGKV1-39, also known as 012)
gene segment, preferably the rearranged germline kappa light chain
IGKV1-39*01/IGKJ1*01 (nomenclature according to the IMGT database,
at [worldwideweb].imgt.org).
[0038] In certain embodiments, the polypeptide chain is rendered
essentially incapable of rearrangement and/or of excluded of any
sequence modification such as normally operating on Ig during the
process of B cell affinity maturation. Therefore, provided is a
transgenic non-human animal that has been provided with an
expression cassette described herein, wherein the rearrangement
and/or sequence modifications are prevented by the absence of
elements at least partially responsible for somatic hypermutation
such as, for example, the MoE.kappa.i enhancer.
[0039] A preferred expression cassette described herein comprises
means for prevention of silencing. In one embodiment, the means for
prevention of silencing are means for insertion into a locus in the
genome of the host cell that is resistant to silencing. The means
for insertion are preferably means for homologous recombination
into the site resistant to silencing. A preferred locus when the
non-human animal is a mouse is the rosa-locus.
[0040] A further preferred expression cassette described herein
comprises in 5'-3' direction: a V.kappa. promoter, a mouse leader,
a human V gene, optionally a MoE.kappa.i enhancer, a rat constant
region (C.kappa.) and optionally a (truncated) MoE.kappa.3'
enhancer.
[0041] Yet a further preferred expression cassette described herein
comprises in 5'-3' direction: a V.kappa. promoter, a human leader,
a human V gene, optionally a MoE.kappa.i enhancer, a rat constant
region (C.kappa.) and optionally a (truncated) MoE.kappa.3'
enhancer.
[0042] Certain antibodies produced as described herein may be used
in human therapeutics and diagnostics. Thus, provided is a method
for producing a desired antibody comprising exposing a non-human
mammal described herein to an antigen such that an antibody
response is induced and isolating the antibodies specific for the
antigen.
[0043] In certain embodiments, provided are methods for producing a
desired antibody comprising exposing a non-human mammal described
herein to an antigen such that an antibody response is induced and
isolating cells producing such antibodies, culturing and optionally
immortalizing the cells and harvesting the antibodies.
[0044] In certain embodiments, provided is a method for producing a
desired antibody comprising exposing a non-human mammal described
herein to an antigen such that an antibody response is induced and
isolating a nucleic acid encoding at least part of such an
antibody, inserting the nucleic acid or a copy or a derivative
thereof in an expression cassette and expressing the antibody in a
host cell.
[0045] The methods for producing antibodies from transgenic mice
are known to a person skilled in the art. Particularly preferred
are methods for production of mixtures of antibodies from one cell,
whereby the nucleic acids encoding these antibodies have been
derived from mice described herein.
[0046] These so-called oligoclonics are disclosed in WO04106375 and
WO05068622, which are incorporated herein by reference.
[0047] Described herein are transgenic non-human mammals,
preferably mice, capable of generating specific and high affinity
hybrid mouse-human antibodies with preferably human immunoglobulin
light chain variable (VL) regions in or near germline configuration
and preferably murine immunoglobulin heavy chain variable (VH)
regions that may have accumulated somatic mutations during the
process of antigen-driven affinity maturation. It is envisaged that
the murine VH regions of the hybrid antibodies may be subjected to
humanization procedures to yield mAbs that have reduced
immunogenicity when applied in humans based on germline or
near-germline VL regions and murine VH regions that have been
humanized.
[0048] In particular, we have shown that transgenic mice that
harbor a DNA expression construct that encodes a rearranged human
VL region under the control of cis-acting genetic elements that
provide timely and regulated expression of the transgene on a
significant proportion of B cells during B cell development, yet
lack elements that direct the somatic hypermutation machinery to
the transgene, are capable of generating specific and high affinity
mouse-human hybrid antibodies with essentially unmutated L chains.
It is shown that the rearranged human transgene is capable of
pairing with a diversity of endogenous murine immunoglobulin H
chains to form mouse-human hybrid immunoglobulins expressed on the
surface of B cells and to sufficiently facilitate murine B cell
development to obtain a sizeable and diverse peripheral B cell
compartment.
[0049] In certain embodiments, the transgene expression construct
harbors the coding sequences of a human rearranged L chain V region
under the control of a human VL promoter to direct B-cell specific
expression. In addition, the construct harbors the murine 3' Ck
enhancer sequence for B cell specific and inducible and high level
expression of the transgene. Furthermore, the construct is designed
to lack regulatory elements that facilitate the recruitment of the
somatic hypermutation machinery to the transgene, such as the
intron enhancer and the 3' C-kappa enhancer.
[0050] In a related embodiment, the rearranged human VL gene is
inserted in the murine Rosa26 locus by site-specific integration.
The Rosa26 locus is useful in the context of the "targeted
transgenesis" approach for efficient generation of transgenic
organisms (such as mice) with a predictable transgene expression
pattern.
[0051] In certain embodiments, the rearranged human VL region is
selected for its capacity to pair with many different murine VH
genes so as to ensure the generation of a population of B cells
with a diverse VH gene repertoire. A method of obtaining such VL
regions comprises amplifying a repertoire of rearranged VH genes
from the B cells of mice and a repertoire of human rearranged
germline VL regions from the B cells of humans and cloning them
into phagemid display vectors to prepare diverse libraries of
hybrid immunoglobulins in bacteria. By nucleotide sequence analysis
of collections of unselected and antigen-selected VH/VL pairs,
human germline VL genes that pair with many different murine VH
genes are identified. A collection of human germline VL genes with
this capacity is described.
[0052] In one embodiment, it is shown that upon immunization with
antigen, the B cells are capable of mounting an immune response,
leading to the generation of B cells that secrete hybrid antibodies
with high specificity and affinity. The V regions encoding these
antibodies are characterized by the human transgenic light chain
that harbors no or very few mutations and a murine heavy chain that
harbors a variable number of mutations introduced by the somatic
hypermutation machinery.
[0053] In a related embodiment, strategies to obtain high affinity
hybrid monoclonal antibodies from the transgenic mice by hybridoma
and display technologies are contemplated as well as procedures to
humanize the murine VH regions to obtain less immunogenic
antibodies for application in humans.
[0054] In one embodiment, provided is an immunoglobulin L chain
transgene construct comprising DNA sequences that encode a human
immunoglobulin VL region in combination with a light chain constant
region (CL) of an animal immunoglobulin protein, which sequences
are operably linked to transcription regulatory sequences that,
when integrated in a non-human transgenic animal, produce an Ig
VL-CL polypeptide with a human VL region that is not or marginally
subject to somatic hypermutation. The Ig VL is capable of pairing
with rearranged VH-CH polypeptides that are generated during B cell
development in the non-human transgenic animal, with the VH-CH
polypeptides retaining the capacity to undergo somatic
hypermutation upon stimulation. The CL region may be of any animal
species and is generally capable of pairing with the CH regions of
the non-human transgenic animal.
[0055] Also included is the use of a transgene construct as above
in producing a transgenic non-human animal capable of the
production of hybrid antibodies consisting of VL-CL polypeptides
and VH-CH polypeptides in which the VL region is of human origin
and the CL, VH and CH may be of any animal species, including
human. Upon immunization, these transgenic animals are capable of
generating high affinity antibodies encoded by somatically
hypermutated VH genes and essentially non-mutated VL genes encoded
by the transgene.
[0056] In another aspect, provided is a process for the production
of a transgenic non-human animal capable of the production of
hybrid antibodies in response to antigenic challenge, comprising
functionally disrupting the endogenous immunoglobulin light chain
locus and inserting into the animal genome a transgene construct of
the invention.
[0057] Included is the use of animals obtainable by this process in
the production of B cells that produce immunoglobulin having human
VL light chain. In another aspect of the invention there is
provided a process for the production of B cells that produce
immunoglobulin having human VL and binding to a selected antigen,
comprising challenging an animal obtainable by a process as above
with the antigen and screening for B cells from the animal that
bind the antigen. Further included is B cells obtainable by this
process and hybridomas obtainable by immortalizing such B cells,
e.g., hybridomas obtained by fusing B cells as above with myeloma
cells. Also included is a process for producing monoclonal antibody
comprising cultivating such a hybridoma. In yet a further aspect,
provided is the use of the above B cells in producing a hybridoma
or corresponding monoclonal antibody.
[0058] Described herein is a process for the production of
immunoglobulin having human VL chain and binding to a selected
antigen, comprising challenging an animal obtainable as above with
the antigen and obtaining immunoglobulin there from.
[0059] In one strategy, as an individual step, a rearranged VL
region encoded by human germline V and J gene segments and a light
chain constant region of any animal species but preferably a murine
constant region is introduced into the mouse germ line. The
transgene DNA may be introduced into the pronuclei of fertilized
oocytes or embryonic stem cells. The integration may be random or
homologous depending on the particular strategy to be employed. For
example, the VL transgene may be introduced by random insertion,
resulting in mice that bear one or multiple copies of the transgene
in the genome. Alternatively, the human VL transgene may be
targeted to a specific genomic locus using site-specific
recombination as described in the art.
[0060] In certain embodiments, the VL transgene is targeted to the
murine ROSA26 locus which is a suitable integration site allowing
strong and predictable expression of inserted transgenes (European
Patent Office document EP 1,439,234 A1, the contents of which are
incorporated herein by this reference). The targeting vector allows
insertion of a single copy of a gene expression cassette, thus
avoiding modulation of transgene expression by the arrangement of
multiple copies. By choosing the autosomal Rosa26 locus as
insertion site, the expression pattern of the inserted transgene in
the non-human animal is predictable. Furthermore, random X
inactivation and/or modulation by chromosomal position effects are
avoided. This also eliminates the need to generate and analyze
multiple transgenic strains for any given transgene. Finally, the
Rosa26 targeting vector for the site-specific integration can be
used for multiple gene expression cassettes. Thus, it may be
envisaged that two or more different rearranged germline human VL
regions are inserted into the Rosa26 locus to further increase the
diversity of the repertoire of hybrid or human antibodies.
[0061] In another embodiment, a rearranged human VL region may be
targeted to the murine Ig kappa or lambda light chain locus so as
to functionally inactivate the endogenous locus or mice containing
the rearranged human VL region may be bred with mice that lack
functional kappa or lambda Ig loci or both. Thus, by using
transformation, using repetitive steps or in combination with
breeding, transgenic animals may be obtained which are able to
produce antibodies harboring the human VL transgene in the
substantial absence of endogenous host immunoglobulin light
chains.
[0062] In one embodiment, a human VL transgene is selected for its
capacity to pair with a substantial portion of murine VH regions to
form a diverse repertoire of functional mouse-human hybrid
antibodies expressed on the surface of B cells. By a substantial
portion of murine VH regions is meant that the human VL pairs with
at least with 0.1% of the murine VH regions generated during B cell
development, more preferably with at least 1% and most preferably
with at least 10%. Methods to identify human VL genes with this
characteristic include randomly pairing a repertoire of human VL
regions with a repertoire of murine VH regions, co-expression of VH
and VL regions in appropriate eukaryotic or prokaryotic expression
vectors and screening for human VL regions that pair with a
substantial portion of murine VH regions. In one embodiment,
phagemid vectors may be used to direct expression of mouse-human
antibody fragments in bacterial cells or to the surface of
filamentous phage and analysis of binding capacity of antibody
fragments by methods known in the art.
[0063] In another embodiment, a human VL transgene is selected for
its capacity to pair with a substantial portion of human VH regions
to form a diverse repertoire of human antibodies expressed on the
surface of B cells. By a substantial portion of human VH regions is
meant that the human VL pairs with at least with 0.1% of the human
VH regions generated during B cell development, more preferably
with at least 1% and most preferably with at least 10%.
[0064] In the latter embodiment, the human VL transgenic mice are
crossed with mice that harbor functional rearranged or
non-rearranged human H chain immunoglobulin loci and functionally
inactivated endogenous H chain Ig loci as described in the art. The
functional inactivation of the two copies of each of the three host
Ig loci (heavy chain, kappa and lambda light chain), where the host
contains the human IgH and the rearranged human VL transgene would
allow for the production of purely human antibody molecules without
the production of host or host human chimeric antibodies. Such a
host strain, by immunization with specific antigens, would respond
by the production of mouse B-cells producing specific human
antibodies, which B-cells are subsequently fused with mouse myeloma
cells or are immortalized in any other manner for the continuous
stable production of human monoclonal antibodies. Alternatively,
the population of B cells is used as a source of VH regions that
can be obtained by constructing cDNA libraries or by PCR
amplification using primers for human VH regions as is known in the
art.
[0065] A human rearranged VL gene is reconstructed in an
appropriate eukaryotic or prokaryotic microorganism and the
resulting DNA fragments can be introduced into pronuclei of
fertilized mouse oocytes or embryonic stem cells. Various
constructs that direct B cell specific expression of VL transgenes
have been described in the art and have the following general
format: a leader sequence and relevant upstream sequences to direct
B cell specific expression of the transgene, a coding sequence of a
human VL transgene, an enhancer sequence that directs B cell
specific and high level expression of the transgene and a murine
constant region gene. In a preferred format, the enhancer is the
C-kappa 3' enhancer because it directs high level expression in
B-lineage cells, but does not recruit somatic hypermutation when
used in transgene constructs.
[0066] In one embodiment, animals, preferably mice, comprising one
or multiple copies of the transgene in the genome are isolated and
analyzed for stable expression. Animals are selected that show
stable expression of the transgene over longer periods of time,
preferably in B-cells. If required, different animal lines
comprising independent insertions of one or multiple copies of the
transgene, preferably on different chromosomes, are crossed to
obtain animals with different insertions of one or multiple copies
of the transgene to increase expression of the transgene in
animals, preferably in B-cells.
[0067] Further provided is progeny of a transgenic non-human animal
described herein, the progeny comprising, at least in its B-cell
lineage, a heavy- or light chain encoding sequence together with a
means that renders the sequence resistant to DNA rearrangements
and/or somatic hypermutations.
[0068] Further provided is progeny of a transgenic non-human animal
described herein, the progeny comprising an expression cassette for
the expression of a desired proteinaceous molecule in cells during
a certain stage of development in cells developing into mature B
cells.
[0069] In addition, provided is a cell that is isolated from a
transgenic non-human animal described herein, the cell comprising a
heavy- or light chain encoding sequence together with a means that
renders the sequence resistant to DNA rearrangements and/or somatic
hypermutations. In addition, provided is a cell that is isolated
from a transgenic non-human animal described herein, the cell
comprising an expression cassette for the expression of a desired
proteinaceous molecule in cells during a certain stage of
development in cells developing into mature B cells. A cell
described herein, preferably an antibody-producing B-cell or a cell
that is capable of differentiating or maturating into an
antibody-producing B-cell, can be used for in vitro production of
antibodies, as is known to the skilled person, for example, from
Gascan et al. 1991, J. Exp. Med. 173:747-750. Methods for
immortalization of a cell described herein are known in the art and
include the generation of hybridomas, for example, by fusion with a
myeloma cell, transformation with Epstein Barr Virus; expression of
the signal transducer of activation and transcription (STAT),
activation via CD40 and IL4 receptor signaling, and/or expression
of Bcl6 (Shvarts et al. 2002, Genes Dev. 16: 681-686).
[0070] In a separate step, the mouse endogenous Kappa and Lambda
light chain loci are rendered essentially non-functional such that
at least the majority of B cells in the transgenic mice bear Ig
receptors that contain the transgenic human VL region. Inactivation
of the endogenous mouse immunoglobulin loci is achieved by targeted
disruption of the appropriate loci by homologous recombination in
mouse embryonic stem cells. The targeted disruption comprises
alteration of the genomic sequence such that substantially no
functional endogenous mouse immunoglobulin Kappa and/or Lambda
light chain is produced. The term "substantially no functional
endogenous mouse immunoglobulin" indicates that the endogenous
Kappa and/or Lambda light chain loci are functionally silenced such
that the level of functional protein expression of the endogenous
Kappa and/or Lambda light chain loci, preferably the endogenous
Kappa light chain locus, is reduced to about 20% of the level of
expression in a reference mouse, more preferred to about 10%, more
preferred to about 5%, more preferred to about 2% and more
preferred to about 1%. In a most preferred embodiment, the level of
functional protein expression of the endogenous Kappa and/or Lambda
light chain loci is reduced to 0%. The level of functional protein
expression can be determined by means known to the skilled person,
including western blotting and pairing with a mouse heavy chain.
The reference mouse is a mouse in which the endogenous Kappa and/or
Lambda light chain loci is not disrupted. The alteration comprises
mutation and/or deletion of gene sequences that are required for
functional expression of the endogenous immunoglobulin genes.
Alternatively, the alteration comprises insertion of a nucleic acid
into the endogenous mouse immunoglobulin Kappa and/or Lambda light
chain loci such that the functional expression of the endogenous
immunoglobulin genes is reduced. In one embodiment, the nucleic
acid comprises a silencing element resulting in transcriptional
silencing of the endogenous immunoglobulin gene. In a further
embodiment, or in addition, the nucleic acid comprises a sequence
that disrupts splicing and/or translation of the endogenous
immunoglobulin gene, for example, by introducing an exon that
renders a frame shift in the coding sequence, or that comprises a
premature stop codon. In each case chimeric animals are generated
which are derived in part from the modified embryonic stem cells
and are capable of transmitting the genetic modifications through
the germ line. The mating of mouse strains with human
immunoglobulin loci to strains with inactivated mouse loci yields
animals which produce antibodies comprising essentially only human
light chains.
[0071] A construct for homologous recombination is prepared by
means known in the art and any undesirable sequences are removed,
e.g., procaryotic sequences. Any convenient technique for
introducing a construct for homologous recombination into a target
cell may be employed. These techniques include spheroplast fusion,
lipofection, electroporation, calcium phosphate-mediated DNA
transfer or direct microinjection. After transformation or
transfection of the target cells, target cells are selected by
means of positive and/or negative markers, for example, by neomycin
resistance and/or acyclovir and/or gancyclovir resistance. Those
cells which show the desired phenotype may then be further analyzed
by restriction analysis, electrophoresis, Southern analysis, PCR,
or the like. By identifying fragments which show the presence of
the lesion(s) at the target locus, cells in which homologous
recombination has occurred to inactivate a copy of the target locus
are identified.
[0072] Furthermore, it is shown that upon immunization, the murine
and human VH regions in the afore-mentioned transgenic mice but not
the VL regions are capable of undergoing somatic hypermutations to
generate high affinity antibodies. Advantageously, these antibodies
encoded by germline VL regions are predicted to contribute to lower
immunogenicity when applied in humans and result in more stable
antibodies that are less prone to aggregation and thus safer for
therapeutic use in humans.
[0073] MAbs derived from the afore-mentioned non-human transgenic
animals or cells all share the same identical human VL regions. It
has been described that mAbs that share the same identical VL
region may be co-expressed in a single clonal cell for the
production of mixtures of recombinant antibodies with functional
binding sites (see the incorpoarated WO04106375 and WO05068622).
Thus, provided is a platform for the generation of specific and
high affinity mAbs that constitute the basis for mixtures of mAbs
produced by clonal cells.
[0074] It is preferred that mAbs derived from the afore-mentioned
non-human transgenic animals or cells are directed against cellular
targets. Preferred targets are human surface-expressed or soluble
proteins or carbohydrate molecules. Further preferred targets are
surface-expressed proteins or carbohydrate molecules that are
expressed on the surface of bacteria, viruses, and other pathogens,
especially of humans.
[0075] More specifically, preferred targets include cytokines and
chemokines, including but not limited to InterLeukin 1beta
(IL1beta), IL2, IL4, IL5, IL7, IL8, IL12, IL13, IL15, IL18, IL21,
IL23 and chemokines such as, for example, CXC chemokines, CC
chemokines, C chemokines (or .gamma. chemokines) such as XCL1
(lymphotactin-.alpha.) and XCL2 (lymphotactin-.beta.), and CX3C
chemokines. Further included as preferred targets are receptor
molecules of the cytokines and chemokines, including type I
cytokine receptors such as, for example, the IL-2 receptor, type II
cytokine receptors such as, for example, interferon receptors,
immunoglobulin (Ig) superfamily receptors, tumor necrosis factor
receptor family including receptors for CD40, CD27 and CD30,
serine/threonine-protein kinase receptors such as TGF beta
receptors, G-protein coupled receptors such as CXCR1-CXCR7, and
tyrosine kinase receptors such as fibroblast growth factor receptor
(FGFR) family members, EGF receptor family members including erbB1
(EGF-R; HER1), erbB2, (HER2), erbB3 (HER3), and erbB4 (HER4),
insulin receptor family members including IGF-R1 and IGF-RII, PDGF
receptor family members, Hepatocyte growth factor receptor family
members including c-Met (HGF-R), Trk receptor family members, AXL
receptor family members, LTK receptor family members, TIE receptor
family members, ROR receptor family members, DDR receptor family
members, KLG receptor family members, RYK receptor family members,
MuSK receptor family members, and vascular endothelial growth
factor receptor (VEGFR) family members.
[0076] Further preferred targets are targets that are
over-expressed or selectively expressed in tumors such as, for
example, VEGF, CD20, CD38, CD33, CEA, EpCAM, PSMA, CD54, Lewis Y,
CD52, CD40, CD22, CD51/CD61, CD74, MUC-1, CD38, CD19, CD262
(TRAIL-R2), RANKL, CTLA4, and CD30; targets that are involved in
chronic inflammation such as, for example, CD25, CD11a, TNF, CD4,
CD80, CD23, CD3, CD14, IFNgamma, CD40L, LD50, CD122, TGFbeta and
TGFalpha.
[0077] Preferred surface-expressed proteins or carbohydrate
molecules that are expressed on the surface of bacteria, viruses,
and other parasitic pathogens, especially of humans, include
surface markers of influenza A and B viruses such as hemagglutinin
(HA) and neuraminidase (NA), filoviruses such as Ebola virus,
rabies, measles, rubella, mumps, flaviviruses such as Dengue virus
types 1-4, tick-borne encephalitis virus, West Nile virus, Japanese
encephalitis virus, and Yellow fever virus, Paramyxoviruses
including Paramyxovirus such as Parainfluenza 1, 3, Rubulavirus
such as Mumpsvirus and Parainfluenza 2, 4, Morbillivirus, and
Pneumovirus such as Respiratory syncytial virus, Vaccinia, small
pox, coronaviruses, including Severe Acute Respiratory Syndrome
(SARS) virus, hepatitis virus A, B and C, Human Immunodeficiency
Virus, Herpes viruses, including cytomegalovirus, Epstein Barr
virus, Herpes simplex virus, and Varicella zoster virus,
parvoviruses such as, for example, B19; Legionella pneumophila;
Listeria monocytogenes; Campylobacter jejuni; Staphylococcus
aureus; E. coli O157:H7; Borrelia burgdorferi; Helicobacter pylori;
Ehrlichia chaffeensis; Clostridium difficile; Vibrio cholera;
Salmonella enterica Serotype Typhimurium; Bartonella henselae;
Streptococcus pyogenes (Group A Strep); Streptococcus agalactiae
(Group B Strep); Multiple drug resistant S. aureus (e.g., MRSA);
Chlamydia pneumoniae; Clostridium botulinum; Vibrio vulnificus;
Parachlamydia pneumonia; Corynebacterium amycolatum; Klebsiella
pneumonia; Linezolid-resistant enterococci (E. faecalis and E.
faecium); and Multiple drug resistant Acinetobacter baumannii.
[0078] Most preferred targets are IL-6 and its receptor,
IL-6Ralpha, glycoprotein-denominated gp130, RSV, especially the
surface proteins F, G and SH and non-structural proteins such as N
and M, and receptor tyrosine kinases, in particular erbB1 (EGF-R;
HER1), erbB2, (HER2), erbB3 (HER3), erbB4 (HER4), IGF-R1 and
IGF-RII, c-Met (HGF-R).
[0079] Therefore, provided is a platform for the generation of
specific and high affinity mAbs against the above mentioned targets
that constitute the basis for mixtures of mAbs produced by clonal
cells. In certain embodiments, the specific and high affinity mAbs
comprise mAbs that are directed against different epitopes on at
least one of the targets. In a further preferred embodiment, the
specific and high affinity mAbs comprise mAbs that are directed
against different targets, such as, for example, one or more
members of the EGF-receptor family, including erbB1 (EGF-R; HER1),
erbB2, (HER2), erbB3 (HER3) and erbB4 (HER4).
[0080] Unless otherwise defined, scientific and technical terms
used in connection with the present invention shall have the
meanings that are commonly understood by those of ordinary skill in
the art. Further, unless otherwise required by context, singular
terms shall include pluralities and plural terms shall include the
singular. Generally, nomenclatures utilized in connection with, and
techniques of, cell and tissue culture, molecular biology, and
protein and oligo- or polynucleotide chemistry and hybridization
described herein are those well known and commonly used in the art.
Standard techniques are used for recombinant DNA, oligonucleotide
synthesis, and tissue culture and transformation (e.g.,
electroporation, lipofection). Enzymatic reactions and purification
techniques are performed according to manufacturer's specifications
or as commonly accomplished in the art or as described herein. The
foregoing techniques and procedures are generally performed
according to conventional methods well known in the art and as
described in various general and more specific references that are
cited and discussed throughout the present specification. See,
e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd
edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. (2001)), which is incorporated herein by reference. The
nomenclatures utilized in connection with, and the laboratory
procedures and techniques of, analytical chemistry, synthetic
organic chemistry, and medicinal and pharmaceutical chemistry
described herein are those well known and commonly used in the art.
Standard techniques are used for chemical syntheses, chemical
analyses, pharmaceutical preparation, formulation, and delivery,
and treatment of patients.
DESCRIPTION OF THE DRAWINGS
[0081] FIG. 1: A topology map of the annealing locations of mouse
specific VH primers and the position of required restriction sites
that are introduced by overhanging sequences at the 3' end of
primers.
[0082] FIG. 2: PCR amplification steps (Amplification, Intermediate
and Site introduction). The location and names of the mouse VH
amplification primers (and mixtures of primers) are indicated per
step.
[0083] FIG. 3: Topology of the MV1043 vector. This vector is used
for the cloning of human or murine VH fragments. O12 (IGKV1-39) is
indicated as the VL gene. Products of this vector in combination
with helper phages in E. coli cells allow the generation of phages
that display Fab fragments on the surface of the phage particles as
a fusion product to the g3 protein and presence of the vector in
the phage as the genetic content (F1 ORI).
[0084] FIG. 4: The topology of the mouse Ckappa locus downstream of
the J-segments. Both enhancers and Ckappa region are indicated. The
lower arrow indicates the region that is removed in order to
silence the locus.
[0085] FIG. 5: The topology of the mouse C-lambda locus. All three
active V-regions are indicated (Igl-V1, V2 and V3) as are the
J-segments (Igl-J1, Igl-J2, Igl-J3, Igl-J4 and the pseudo segment
Igl-J3p) and constant regions (Igl-C1, Igl-C2, Igl-C3 and Igl-C4).
The regions that are deleted in order to silence the locus are
indicated by deletion markers. These deletions include all active V
genes (1, 2 and 3) and the intergenic segment between V2 and
V3.
[0086] FIG. 6: Construct topology of IGKV1-39/J-Ck with an intron
located in the leader open reading frame (ORF).
[0087] FIG. 7: Construct topology of IGLV2-14/J-Ck with an intron
located in the leader open reading frame (ORF).
[0088] FIG. 8: Construct topology of VkP-IGKV1-39/J-Ck (VkP-O12).
The promoter originates from the IGKV1-39 gene and is placed
directly in front of the required elements for efficient
transcription and translation. Intergenic sequences (including the
enhancers) are derived from mice and obtained from BAC clones. The
C-kappa sequence codes for the kappa constant region of rat.
[0089] FIG. 9: Construct topology of VkP-IGLV2-14/J-Ck (VkP-2a2).
The promoter originates from the IGKV1-39 gene and is placed
directly in front of the required elements for efficient
transcription and translation. Intergenic sequences (including the
enhancers) are derived from mice and obtained from BAC clones. The
C-kappa sequence codes for the kappa constant region of rat.
[0090] FIG. 10: Construct topology of VkP-IGKV1-39/J-Ck-.DELTA.1
(VkP-O12-del1) is identical to VkP-IGKV1-39/J-Ck from FIG. 9 except
that the intron enhancer region is removed.
[0091] FIG. 11: Construct topology of VkP-IGKV1-39/J-Ck-.DELTA.2
VkP-O12-del2) is identical to VkP-IGKV1-39/J-Ck-.DELTA.1 from FIG.
10 except that a large piece of the intergenic region between the
Ck gene and 3' enhancer is deleted. In addition, the 3' enhancer is
reduced in size from 809 bp to 125 bp.
[0092] FIG. 12: Overview of the sequences used or referred to in
this application: Human germline IGKV1-39/J DNA (SEQ ID NO:84);
human germline IGKV1-39/J Protein (SEQ ID NO:85); human germline
IGLV2-14/J DNA (SEQ ID NO:86); human germline IGLV2-14/J Protein
(SEQ ID NO:87); Rat IGCK allele a DNA (SEQ ID NO:88); Rat IGCK
allele a protein (SEQ ID NO:89); IGKV1-39/J-Ck (SEQ ID NO:90);
IGLV2-14/J-Ck (SEQ ID NO:91); VkP-IGKV1-39/J-Ck (SEQ ID NO:92);
VkP-IGKV1-39/J-Ck-.DELTA.1 (SEQ ID NO:93);
VkP-IGKV1-39/J-Ck-.DELTA.2 (SEQ ID NO:94); VkP-IGLV2-14/J-Ck (SEQ
ID NO:95); pSELECT-IGKV1-39/J-Ck (SEQ ID NO:96);
pSelect-IGLV2-14/J-Ck (SEQ ID NO:97); MV1043 (SEQ ID NO:98); and
MV1057 (SEQ ID NO:99).
[0093] FIGS. 13A-C: Generation of Rosa26-IgVk1-39 KI allele. FIG.
13A Schematic drawing of the pCAGGS-IgVK1-39 targeting vector. FIG.
13B Nucleotide sequence of the pCAGGS-IgVK1-39 targeting vector
(SEQ ID NO:100). FIG. 13C Targeting strategy.
[0094] FIGS. 14A-C: FIG. 14A Southern blot analysis of genomic DNA
of ES clones comprising an insertion of the pCAGGS-IgVK1-39
targeting vector. Genomic DNA of four independent clones was
digested with AseI and probed with 5e1 indicating the 5'-border of
the targeting vector. All clones comprise a correct insertion of
the targeting vector at the 5' end. FIG. 14B Southern blot analysis
of genomic DNA of ES clones comprising an insertion of the
pCAGGS-IgVK1-39 targeting vector. Genomic DNA of four independent
clones was digested with MscI and probed with 3e1 indicating the
3'-border of the targeting vector. All clones comprise a correct
insertion of the targeting vector at the 3' end. FIG. 14C Southern
blot analysis of genomic DNA of ES clones comprising an insertion
of the pCAGGS-IgVK1-39 targeting vector. Genomic DNA of four
independent clones was digested with BamHI and probed with an
internal Neo probe indicating the 5'-border of the targeting
vector. All clones comprise a correct, single insertion of the
targeting vector.
[0095] FIGS. 15A-C: Generation of Rosa26-IgVl2-14 KI allele. FIG.
15A Schematic drawing of the pCAGGS-IgVL2-14 targeting vector. FIG.
15B Nucleotide sequence of the pCAGGS-IgVL2-14 targeting vector
containing the CAGGS expression insert (SEQ ID NO:101) based on the
rearranged germline IGLV2-14/J V lambda region (IGLV2-14/J-Ck).
FIG. 15C Targeting strategy.
[0096] FIGS. 16A-C: Epibase.RTM. profile of IGKV1-39 residues 1-107
(SEQ ID NO:85). FIG. 16A displays the binding strength for DRB1
allotypes, while FIG. 16C displays the binding strength for
DRB3/4/5, DQ and DP allotypes. The values in the figure represent
dissociation constants (Kds) and are plotted on a logarithmic scale
in the range 0.01 .mu.M-0.1 .mu.M (very strong binders may have run
off the plot). For medium binding peptides, qualitative values are
given only, and weak and non-binders are not shown. Values are
plotted on the first residue of the peptide in the target sequence
(the peptide itself extends by another nine residues). Importantly,
only the strongest binding receptor for each peptide is shown:
cross-reacting allotypes with lower affinity are not visible in
this plot. The strongest binding receptor is indicated by its
serotypic name. Finally, any germline-filtered peptides are plotted
with a lighter color in the epitope map (in this case, no non-self
epitopes were found). FIG. 16B shows the HLA binding promiscuity
for every decameric peptide (Y-axis: the number of HLA allotypes
recognizing critical epitopes in each of the peptides starting at
the indicated residue shown on the X-axis). The promiscuity is
measured as the number of allotypes out of the total of 47 for
which the peptide is a critical binder. White columns refer to
self-peptides, and black columns (absent here) to non-self
peptides.
[0097] FIG. 17: Epitope map of IGKV1-39 showing the presence of
peptide binders predicted in the sequence of IGKV1-39 by serotype
in the 15-mer format. Each 15-mer is numbered as indicated in the
top of the figure. The full sequence of the corresponding 15-mer is
listed in Table 7. Black boxes indicate the presence of one or more
critical self-epitopes in the 15-mer for the serotype listed on the
left. Critical epitopes are operationally defined as strong or
medium DRB1 binders and strong DRB3/4/5 or DP or DQ binders.
[0098] FIGS. 18A-B: Constitutive knock-out (KO) of the Ig kappa
locus. FIG. 18A Targeting strategy. FIG. 18B Schematic drawing of
the pIgKappa targeting vector.
[0099] FIGS. 19A-B: Constitutive KO of the Ig lambda locus. FIG.
19A First step of the targeting strategy. FIG. 19B Second step of
the targeting strategy.
[0100] FIGS. 20A-C: Schematic drawing of targeting vectors. FIG.
20A pVkP-O12 (VkP-IGKV1-39/J-Ck); FIG. 20B pVkP-O12-del1
(VkP-IGKV1-39/J-Ck-.DELTA.1); FIG. 20C pVkP-O12-del2
(VkP-IGKV1-39/J-Ck-.DELTA.2).
[0101] FIGS. 21A-C: Targeting strategies for insertion of transgene
into the Rosa26 locus by targeted transgenesis using RMCE. FIG. 21A
VkP-O12 (VkP-IGKV1-39/J-Ck); FIG. 21B VkP-O12-del1
(VkP-IGKV1-39/J-Ck-.DELTA.1); FIG. 21C VkP-O12-del2
(VkP-IGKV1-39/J-Ck-.DELTA.2).
[0102] FIG. 22: Topology of the MV 1057 vector. Replacing the
indicated stuffer fragment with a VH fragment yields an expression
vector that can be transfected to eukaryotic cells for the
production of IgG1 antibodies with light chains containing an O12
(IGKV1-39) VL gene.
[0103] FIG. 23: Lack of transgenic human Vk1 light chain expression
in non-B cell populations of the spleen.
[0104] FIG. 24: Transgenic human Vk1 light chain is expressed in
all B cell populations of the spleen.
[0105] FIG. 25: Transgenic human Vk1 light chain is expressed in B1
cells of the peritoneal cavity.
[0106] FIGS. 26A-B: Transgenic human Vk1 light chain is not
expressed in pro- and pre-B cells but in the immature and
recirculating populations B cells in the bone marrow. FIG. 26A
Gating of bone marrow cells. FIG. 26B Histograms of transgene
expression with overlay from one WT control.
[0107] FIG. 27: Transgenic human Vk1 light chain is directly
correlated with endogenous light chain and IgM expression in
circulating B cells in the blood.
DETAILED DESCRIPTION OF THE INVENTION
Examples
Example 1
Human Light Chain V-Gene Clones
[0108] This example describes the rationale behind the choice of
two human light chain V-genes, one gene of the kappa type and one
gene of the lambda type, that are used as a proof of concept for
light chain expressing transgenic mice. De Wildt et al. 1999 (de
Wildt et al. (1999), J. Mol. Biol. 285(3):895) analyzed the
expression of human light chains in peripheral IgG-positive
B-cells. Based on these data, IGKV1-39 (O12) and IGLV2-14 (2a2)
were chosen as light chains as they were well represented in the
B-cell repertoire. The J-segment sequence of the light chains has
been chosen based upon sequences as presented in GenBank ABA26122
for IGKV1-39 (B. J. Rabquer, S. L. Smithson, A. K. Shriner and M.
A. J. Westerink) and GenBank AAF20450 for IGLV2-14 (O. Ignatovich,
I. M. Tomlinson, A. V. Popov, M. Bruggemann and G. J. Winter, J.
Mol. Biol. 294 (2):457-465 (1999)).
[0109] All framework segments are converted into germline amino
acid sequences to provide the lowest immunogenicity possible in
potential clinical applications.
Example 2
Obtaining Mouse Heavy Chain V-Genes that Pair with Human IGKV1-39
Gene Segment to Form Functional Antibody Binding Sites
[0110] This example describes the identification of mouse heavy
chain V-genes that are capable of pairing with a single, rearranged
human germline IGKV1-39/J region. A spleen VH repertoire from mice
that were immunized with tetanus toxoid was cloned in a phage
display Fab vector with a single human IGKV1-39-C kappa light chain
and subjected to panning against tetanus toxoid. Clones obtained
after a single round of panning were analyzed for their binding
specificity. The murine VH genes encoding tetanus toxoid-specific
Fab fragments were subjected to sequence analysis to identify
unique clones and assign VH, DH and JH utilization.
[0111] Many of the protocols described here are standard protocols
for the construction of phage display libraries and the panning of
phages for binding to an antigen of interest and described in
Antibody Phage Display: Methods and Protocols (editor(s): Philippa
M. O'Brien and Robert Aitken).
Immunizations
[0112] BALB/c mice received one immunization with tetanus toxoid
and were boosted after six weeks with tetanus toxoid.
Splenocyte Isolation
[0113] Preparation of spleen cell suspension. After dissection, the
spleen was washed with PBS and transferred to a 60 mm Petri dish
with 20 ml PBS. A syringe capped with 20 ml PBS and a G20 needle
was used to repeatedly flush the spleen. After washing the flushed
cells with PBS, the cells were carefully brought into suspension
using 20 ml PBS and left on a bench for five minutes to separate
the splenocytes from the debris and cell clusters. The splenocytes
suspension was transferred on top of a Ficoll-Paque.TM. PLUS-filled
tube and processed according to the manufacturer's procedures for
lymphocyte isolation (Amersham Biosciences).
RNA Isolation and cDNA Synthesis
[0114] After isolation and pelleting of lymphocytes, the cells were
suspended in TRIzol LS Reagent (Invitrogen) for the isolation of
total RNA according to the accompanying manufacturer's protocol and
subjected to reverse transcription reaction using 1 microgram of
RNA, Superscript III RT in combination with dT20 according to
manufacturer's procedures (Invitrogen).
PCR Amplification of cDNA
[0115] The cDNA was amplified in a PCR reaction using primer
combinations that allow the amplification of approximately 110
different murine V-genes belonging to 15 VH families (Table 1;
RefSeq NG.sub.--005838; Thiebe et al. 1999, European Journal of
Immunology 29:2072-2081). In the first round, primer combinations
that bind to the 5' end of the V-genes and 3' end of the J regions
were used. In the second round, PCR products that were generated
with the MJH-Rev2 primer were amplified in order to introduce
modifications in the 3' region to enable efficient cloning of the
products. In the last round of amplification, all PCR products were
amplified using primers that introduce a SfiI restriction site at
the 5' end and a BstEII restriction site at the 3' end (see FIGS. 1
and 2, and Table 1).
[0116] Reaction conditions for 1st round PCR: four different
reactions combining all 25 forward primers (MVH1 to MVH25, Table 1
and FIG. 2) and one reverse primer per reaction (MJH-Rev1,
MJH-Rev2, MJH-Rev3 or MJH-Rev4; see Table 1 and FIG. 2). Fifty
microliters PCR volumes were composed of 2 microliters cDNA (from
RT reactions), 10 microliters 5* Phusion polymerase HF buffer, 40
nM of each of the 25 forward primers (total concentration of 1
micromolar), 1 micromolar reverse primer, 1 microliter 10 mM dNTP
stock, 1.25 unit Phusion polymerase and sterile MQ water. The
thermocycler program consisted of a touch down program: one cycle
98.degree. C. for 30 seconds, 30 cycles 98.degree. C. for ten
seconds, 58.degree. C. decreasing 0.2.degree. C. per cycle ten
seconds, 72.degree. C. 20 seconds and one cycle 72.degree. C. for
three minutes. The second round PCR program was set up only for the
products of the first PCR that contain the MJH-Rev2 primer: two
different reactions combining either the ExtMVH-1 or ExtMVH-2
primers (Table 1 and FIG. 2) in combination with the reverse primer
ExtMJH-Rev2int (Table 1 and FIG. 2). Fifty microliters PCR volumes
were composed of 50 ng PCR product (from first PCR round), 10
microliters 5* Phusion polymerase HF buffer, 500 nM of each forward
primer, 1 micromolar reverse primer, 1 microliter 10 mM dNTP stock,
1.25 unit Phusion polymerase and sterile MQ water. The thermocycler
program consisted of a touch down program followed by a regular
amplification step: one cycle 98.degree. C. for 30 seconds, ten
cycles 98.degree. C. for ten seconds, 65.degree. C. decreasing
1.5.degree. C. per cycle ten seconds, 72.degree. C. 20 seconds, ten
cycles 98.degree. C. for ten seconds, 55.degree. C. ten seconds,
72.degree. C. 20 seconds and one cycle 72.degree. C. for three
minutes. The third round PCR program was setup as described in FIG.
2. Fifty microliters PCR volumes were composed of 50 ng PCR product
(from earlier PCR rounds, FIG. 2), 10 microliters 5* Phusion
polymerase HF buffer, 1 micromolar forward primer (Table 1 and FIG.
2), 1 micromolar reverse primer, 1 microliter 10 mM dNTP stock,
1.25 unit Phusion polymerase and sterile MQ water. The program
consists of a touch down program followed by a regular
amplification step: one cycle 98.degree. C. for 30 seconds, ten
cycles 98.degree. C. for ten seconds, 65.degree. C. decreasing
1.5.degree. C. per cycle ten seconds, 72.degree. C. 20 seconds, ten
cycles 98.degree. C. for ten seconds, 55.degree. C. ten seconds,
72.degree. C. 20 seconds and one cycle 72.degree. C. for three
minutes. After PCR amplifications, all PCR products were gel
purified using Qiaex II according to the manufacturer's
protocols.
Restriction Enzyme Digestions
[0117] Purified products were digested with BstEII and SfiI in two
steps. First 1 microgram of DNA was digested in 100 microliters
reactions consisting of 10 microliters of 10* NEB buffer 3 (New
England Biolabs), 1 microliter 100* BSA, 12.5 unit BstEII and
sterile water for six hours at 60.degree. C. in a stove. The
products were purified using Qiaquick PCR Purification kit from
Qiagen according to the manual instructions and eluted in 40
microliters water. Next all products were further digested with
SfiI in 100 microliters reactions consisting of 10 microliters of
10* NEB buffer 2 (New England Biolabs), 1 microliter 100* BSA, 12.5
unit SfiI and sterile water for 12 hours at 50.degree. C. in a
stove. The digested fragments were purified by Qiaquick Gel
Extraction kit following gel separation on a 20 cm 1.5% agarose TBE
plus ethidium bromide gel at 80 V. 100 micrograms of the acceptor
vector (MV1043, FIGS. 3 and 12) was digested with 50 units Eco91I
in 600 microliters under standard conditions (Tango buffer) and
next purified on a 0.9% agarose gel. After a second digestion step
under prescribed conditions with 400 units SfiI in 500 microliters
for 12 hours, 100 units BsrGI were added for three hours at
50.degree. C.
Ligations
[0118] Each PCR product was ligated separately according to the
following scheme: 70 ng digested PCR products, 300 ng digested
acceptor vector, 100 units T4 Ligase (NEB), 1* ligase buffer in 30
microliters for 16 hours at 12.degree. C. The ligation reactions
were purified with phenol/chloroform/isoamyl alcohol extractions
followed by glycogen precipitations (Sigma Aldrich #G1767)
according to the manufacturer's protocol and finally dissolved in
25 microliters sterile water.
Transformations and Library Storage
[0119] The purified ligation products were transformed by
electroporation using 1200 microliters TG1 electrocompetent
bacteria (Stratagene #200123) per ligation batch and plated on LB
carbenicillin plates containing 4% glucose. Libraries were
harvested by scraping the bacteria in 50 ml LB carbenicillin. After
centrifugation at 2000 g for 20 minutes at 4.degree. C., the
bacterial pellets were resuspended carefully in 2 ml ice cold
2*TY/30% glycerol on ice water and frozen on dry ice/ethanol before
storage at -80.degree. C.
Library Amplification
[0120] Libraries were grown and harvested according to procedures
as described by Kramer et al. 2003 (Kramer et al. (2003), Nucleic
Acids Res. 31(11):e59) using VCSM13 (Stratagene) as helper phage
strain.
Selection of Phages on Coated Immunotubes
[0121] Tetanus toxoid was dissolved in PBS in a concentration of 2
.mu.g/ml and coated to MAXISORP.TM. Nunc-Immuno Tube (Nunc 444474)
overnight at 4.degree. C. After discarding the coating solution,
the tubes were blocked with 2% skim milk (ELK) in PBS (blocking
buffer) for one hour at RT. In parallel, 0.5 ml of the phage
library was mixed with 1 ml blocking buffer and incubated for 20
minutes at room temperature. After blocking the phages, the phage
solution was added to the tetanus toxoid-coated tubes and incubated
for two hours at RT on a slowly rotating platform to allow binding.
Next, the tubes were washed ten times with PBS/0.05% TWEEN.TM.-20
detergent followed by phage elution by an incubation with 1 ml 50
mM glycine-HCl pH 2.2 ten minutes at RT on rotating wheel and
directly followed by neutralization of the harvested eluent with
0.5 ml 1 M Tris-HCl pH 7.5.
Harvesting Phage Clones
[0122] Five ml XL1-Blue MRF (Stratagene) culture at O.D. 0.4 was
added to the harvested phage solution and incubated for 30 minutes
at 37.degree. C. without shaking to allow infection of the phages.
Bacteria were plated on Carbenicillin/Tetracycline 4% glucose 2*TY
plates and grown overnight at 37.degree. C.
Phage Production
[0123] Phages were grown and processed as described by Kramer et
al. 2003 (Kramer et al. 2003, Nucleic Acids Res. 31(11):e59) using
VCSM13 as helper phage strain.
Phage ELISA
[0124] ELISA plates were coated with 100 microliters tetanus toxoid
per well at a concentration of 2 micrograms/ml in PBS overnight at
4.degree. C. Plates coated with 100 microliters thyroglobulin at a
concentration of 2 micrograms/ml in PBS were used as a negative
control. Wells were emptied, dried by tapping on a paper towel,
filled completely with PBS-4% skimmed milk (ELK) and incubated for
one hour at room temperature to block the wells. After discarding
the block solution, phage minipreps pre-mixed with 50 .mu.l
blocking solution were added and incubated for one hour at RT. Next
five washing steps with PBS-0.05% Tween-20 removed unbound phages.
Bound phages were detected by incubating the wells with 100
microliters anti-M13-HRP antibody conjugate (diluted 1/5000 in
blocking buffer) for one hour at room temperature. Free antibody
was removed by repeating the washing steps as described above,
followed by TMB substrate incubation until color development was
visible. The reaction was stopped by adding 100 microliters of 2 M
H.sub.2SO.sub.4 per well and analyzed on an ELISA reader at 450 nm
emission wavelength (Table 2). Higher numbers indicate stronger
signals and thus higher incidence of specific binding of the
phage-Fab complex.
Sequencing
[0125] Clones that gave signals at least three times above the
background signal (Table 2) were propagated, used for DNA miniprep
procedures (see procedures Qiagen miniPrep manual) and subjected to
nucleotide sequence analysis. Sequencing was performed according to
the Big Dye 1.1 kit accompanying manual (Applied Biosystems) using
a reverse primer (CH1_Rev1, Table 1) recognizing a 5' sequence of
the CH1 region of the human IgG1 heavy chain (present in the Fab
display vector MV1043, FIGS. 3 and 12). Mouse VH sequences of 28
tetanus toxoid binding clones are depicted in Table 3. The results
show that the selected murine VH genes belong to different gene
families, and different individual members from these gene families
are able to pair with the rearranged human IGKV1-39/J VH region to
form functional tetanus toxoid-specific antibody binding sites.
From the sequence analyses, it was concluded that the murine VH
regions utilize a diversity of DH and JH gene segments.
Example 3
Silencing of the Mouse Kappa Light Chain Locus
[0126] This example describes the silencing of the mouse endogenous
kappa light chain locus. The endogenous kappa locus is modified by
homologous recombination in ES cells, followed by the introduction
of genetically modified ES cells in mouse embryos to obtain
genetically adapted offspring.
[0127] A vector that contains an assembled nucleotide sequence
consisting of a part comprising the J-region to 338 bp downstream
of the J5 gene segment fused to a sequence ending 3' of the 3' CK
enhancer is used for homologous recombination in ES cells. The
assembled sequence is used to delete a genomic DNA fragment
spanning from 3' of the JK region to just 3' of the 3' CK enhancer.
As a consequence of this procedure, the CK constant gene, the 3'
enhancer and some intergenic regions are removed (see FIGS. 4 and
18A-B).
Construction of the Targeting Vector
[0128] A vector that received 4.5-8 kb flanking arms on the 3' and
5' end fused to the deletion segment was used for targeted
homologous recombination in an ES cell line. Both arms were
obtained by PCR means ensuring maximum homology. The targeting
strategy allows generation of constitutive KO allele. The mouse
genomic sequence encompassing the Igk intronic enhancer, Igk
constant region and the Igk 3' enhancer was replaced with a PuroR
cassette, which was flanked by F3 sites and inserted downstream of
the Jk elements. Flp-mediated removal of the selection marker
resulted in a constitutive KO allele. The replacement of the Igk
MiEk-Igk C-Igk 3'E genomic region (approximately 10 kb) with a
F3-Puro cassette (approx. 3 kb) was likely to decrease the
efficiency of homologous recombination. Therefore, the arms of
homology were extended accordingly and more ES cell colonies were
analyzed after transfection in order to identify homologous
recombinant clones.
Generation of ES Cells Bearing the Deleted Kappa Fragment
[0129] The generation of genetically modified ES cells was
essentially performed as described (Seibler et al. (2003), Nucleic
Acids Res. February 15; 31(4):e12). See also Example 14 for a
detailed description.
Generation of ES Mice by Tetraploid Embryo Complementation
[0130] The production of mice by tetraploid embryo complementation
using genetically modified ES cells was essentially performed as
described (Eggan et al., PNAS 98:6209-6214; J. Seibler et al.
(2003), Nucleic Acids Res. February 15; 31(4):e12; Hogan et al.
(1994), Summary of mouse development, Manipulating the Mouse
Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor
N.Y., pp. 253-289).
Example 4
Silencing of the Mouse Lambda Light Chain Locus
[0131] This example describes the silencing of the mouse endogenous
lambda light chain locus. The endogenous lambda locus is modified
by homologous recombination in ES cells followed by the
introduction of genetically modified ES cells in mouse embryos to
obtain genetically adapted offspring.
[0132] Two regions of the murine lambda locus that together contain
all functional lambda V regions are subject to deletion.
[0133] The first region targeted for homologous recombination-based
deletion is a region that is located 408 bp upstream of the start
site of the IGLV2 gene segment and ends 215 bp downstream of IGLV3
gene segment, including the intergenic sequence stretch between
these IGLV gene segments. The second region that is subject to a
deletion involves the IGLV1 gene segment consisting of a fragment
spanning from 392 bp upstream to 171 bp downstream of the IGLV1
gene segment. As a consequence of these two deletion steps, all
functional V-lambda genes segments are deleted, rendering the locus
functionally inactive (FIGS. 5 and 19A-B).
Construction of the Targeting Vectors
[0134] Vectors that received 3-9.6 kb flanking arms on the 3' and
5' end fused to the deletion segment were used for targeted
homologous recombination in an ES cell line. Both arms were
obtained by PCR means ensuring maximum homology. In a first step,
the mouse genomic sequence encompassing the Igl V2-V3 regions were
replaced with a PuroR cassette flanked by F3 sites, which yields a
constitutive KO allele after Flp-mediated removal of selection
marker (see FIG. 19A). In a second step, the mouse genomic sequence
encompassing the Igl V1 region was replaced with a Neo cassette in
ES cell clones which already carried a deletion of the Igl V2-V3
regions (see FIG. 19B). The selection marker (NeoR) was flanked by
FRT sites. A constitutive KO allele was obtained after Flp-mediated
removal of selection markers.
Generation of ES Cells Bearing the Deleted Lambda Fragment
[0135] The generation of genetically modified ES cells was
essentially performed as described (J. Seibler, B. Zevnik, B.
Kuter-Luks, S. Andreas, H. Kern, T. Hennek, A. Rode, C. Heimann, N.
Faust, G. Kauselmann, M. Schoor, R. Jaenisch, K. Rajewsky, R. Kuhn,
F. Schwenk (2003), Nucleic Acids Res., February 15; 31(4):e12). See
also, Example 14 for a detailed description. To show that both
targeting events occurred on the same chromosome several double
targeted clones were selected for the in vitro deletion with pCMV
C31deltaCpG. The clones were expanded under antibiotic pressure on
a mitotically inactivated feeder layer comprised of mouse embryonic
fibroblasts in DMEM High Glucose medium containing 20% FCS (PAN)
and 1200 .mu./mL Leukemia Inhibitory Factor (Millipore ESG 1107).
1.times.10.sup.7 cells from each clone were electroporated with 20
.mu.g of circular pCMV C31deltaCpG at 240 V and 500 .mu.F and
plated on four 10 cm dishes each. Two to three days after
electroporation, cells were harvested and analyzed by PCR. Primers
used were:
TABLE-US-00001 2005_5: (SEQ ID NO: 1) CCCTTTCCAATCTTTATGGG 2005_7:
(SEQ ID NO: 2) AGGTGGATTGGTGTCTTTTTCTC 2005_9: (SEQ ID NO: 3)
GTCATGTCGGCGACCCTACGCC
[0136] PCR reactions were performed in mixtures comprising 5 .mu.l
PCR Buffer 10.times. (Invitrogen), 2 .mu.l MgCl.sub.2 (50 mM), 1
.mu.l dNTPs (10 mM), 1 .mu.l first primer (5 .mu.M), 1 .mu.l second
primer (5 .mu.M), 0.4 .mu.l Taq (5 U/ul, Invitrogen), 37.6 .mu.l
H.sub.2O, and 2 .mu.l DNA. The program used was 95.degree. C. for
five minutes; followed by 35 cycles of 95.degree. C. for 30
seconds; 60.degree. C. for 30 seconds; 72.degree. C. for 1 minute;
followed by 72.degree. C. for ten minutes.
Generation of ES Mice by Tetraploid Embryo Complementation
[0137] The production of mice by tetraploid embryo complementation
using genetically modified ES cells was essentially performed as
described (Eggan et al., PNAS 98:6209-6214; J. Seibler, B. Zevnik,
B. Kuter-Luks, S. Andreas, H. Kern, T. Hennek, A. Rode, C. Heimann,
N. Faust, G. Kauselmann, M. Schoor, R. Jaenisch, K. Rajewsky, R.
Kuhn, and F. Schwenk (2003), Nucleic Acids Res., February 15;
31(4):e12; Hogan et al. (Cold Spring Harbor Laboratory Press, Cold
Spring Harbor N.Y.), pp. 253-289).
Example 5
Construction of the CAGGS Expression Insert Based on a Rearranged
Human Germline IGKV1-39/J-Ck Gene (IGKV1-39/J-Ck)
[0138] This example describes the construction of a CAGGS
expression cassette incorporating the rearranged human germline
IGKV1-39/J region. This insert expression cassette encompasses
cloning sites, a Kozak sequence, a leader sequence containing an
intron, an open reading frame of the rearranged IGKV1-39 region, a
rat CK constant region from allele a and a translational stop
sequence (IGKV1-39/J-Ck; FIG. 6). The primary construct consists of
naturally occurring sequences and has been analyzed and optimized
by removing undesired cis acting elements like internal TATA-boxes,
poly adenylation signals, chi-sites, ribosomal entry sites, AT-rich
or GC-rich sequence stretches, ARE-, INS- and CRS sequence
elements, repeat sequences, RNA secondary structures, (cryptic)
splice donor and acceptor sites and splice branch points (GeneArt
GmbH). In addition, the codon usage in the open reading frame
regions is optimized for expression in mice. The intron sequence is
unchanged and thus represents the sequence identical to the coding
part of the human IGKV1-39 leader intron.
[0139] At the 5' end of the expression cassette, a NotI site was
introduced and on the 3' site a NheI site. Both sites are used for
cloning in the CAGGS expression module. After gene assembly
according to methods used by GeneArt, the insert is digested with
NotI-NheI and cloned into the expression module containing a CAGGS
promoter, a stopper sequence flanked by LoxP sites ("foxed"), a
polyadenylation signal sequence and, at the 5' and 3' end,
sequences to facilitate homologous recombination into the Rosa26
locus of mouse ES cell lines. Promoter and/or cDNA fragments were
amplified by PCR, confirmed by sequencing and/or cloned directly
from delivered plasmids into an RMCE exchange vector harboring the
indicated features. A schematic drawing and the confirmed sequence
of the final targeting vector pCAGGS-IgVK1-39 are shown in FIGS.
13A and 13B. The targeting strategy is depicted in FIG. 13C.
Example 6
CAGGS Expression Insert Based on the Rearranged Germline IGLV2-14/J
V Lambda Region (IGLV2-14/J-Ck)
[0140] This example describes the sequence and insertion of an
expression cassette incorporating the rearranged germline
IGLV2-14/J V lambda region. This insert encompasses cloning sites,
a Kozak sequence, a leader sequence containing an intron, an open
reading frame of the rearranged IGLV2-14/J region, a rat CK
constant region from allele a and a translational stop sequence
(IGLV2-14/J-Ck; FIG. 7). The primary construct consists of
naturally-occurring sequences and has been analyzed and optimized
by removing undesired cis acting elements like: internal
TATA-boxes, poly adenylation signals, chi-sites, ribosomal entry
sites, AT-rich or GC-rich sequence stretches, ARE-, INS- and CRS
sequence elements, repeat sequences, RNA secondary structures,
(cryptic) splice donor and acceptor sites and splice branch points
(GeneArt GmbH). In addition, the codon usage in the open reading
frame regions was optimized for expression in mice. The intron
sequence is unchanged and thus represents the sequence identical to
the human IGKV1-39 leader intron.
[0141] At the 5' end of the expression cassette, a NotI site was
introduced and on the 3' site a NheI site. Both sites are used for
cloning in the CAGGS expression module as described by
TaconicArtemis. After gene assembly according to methods used by
GeneArt, the insert was digested with NotI-NheI and cloned into the
expression module containing a CAGGS promoter, a stopper sequence
flanked by LoxP sites ("foxed"), a polyadenylation signal sequence
and, at the 5' and 3' end, sequences to facilitate homologous
recombination into the Rosa26 locus of mouse ES cell lines. To
construct the final ROSA26 RMCE targeting vector, promoter and/or
cDNA fragments were amplified by PCR. Amplified products were
confirmed by sequencing and/or cloned directly from delivered
plasmids into an RMCE exchange vector harboring the indicated
features. A schematic drawing and the confirmed sequence of the
final targeting vector pCAGGS-IgVL2-14 is shown in FIGS. 15A and
15B. The targeting strategy is depicted in FIG. 15C.
Example 7
Expression of IGKV1-39/J-Ck in HEK293 Cell Lines
(pSELECT-IGKV1-39/J-Ck)
[0142] This example describes a method to verify that the
IGKV1-39/J-Ck constructs described in Example 5 enable expression
and detection of the IGKV1-39/J-Ck L chain in HEK293 cells. The
IGKV1-39/J insert (FIG. 6) was modified at the 5' end by changing
the NotI site into a SalI site. This change is required for cloning
of the product into the expression cassette plasmid pSELECT-hygro
(InvivoGen). The CAGGS expression insert IGKV1-39/J-Ck and
pSELECT-hygro were digested with SalI and NheI, ligated and used to
transform competent XL1-Blue cells using standard techniques.
Colonies were picked and DNA purified using Qiagen Midi-prep
columns according to the manufacturer's procedures. The resulting
light chain (LC) expressing vector named
0817676_pSELECT.sub.--0815426 was used to transfect HEK293 cells
with Fugene6 (Roche) according to the manufacturer's protocols.
Supernatants were screened for the presence of IGKV1-39/J-Ck light
chains by ELISA and western blot using anti-rat-Ck antibodies
(Beckton Dickinson #550336 and 553871) and protocols used in the
art.
[0143] The VH of anti-tetanus toxoid (TT) IgG MG1494 was cloned
into IgG expression vector MV1056 using restriction sites SfiI and
BstEII. The resulting clone was sequence verified. HEK293T cells
were transfected with five different vector combinations as shown
in Table 4 (see Example 8 for details of vector
0817678_pSELECT.sub.--0815427). Supernatants were harvested and IgG
concentrations determined (see Table 4). No IgG could be detected
for supernatants A and B containing light chain only as expected
(detection antibody recognized Fc part of IgG). IgG concentration
in supernatants C and D was comparable to that of positive control
supernatant E, indicating correct expression of the light chain
constructs.
[0144] Binding to TT was analyzed by ELISA to check functionality
of the produced antibodies, using hemoglobin as negative control
antigen. No TT-specific binding could be detected for supernatants
A and B containing light chain only, as expected. TT-specific
binding for supernatants C and D was at least as good as for
positive control supernatant E, confirming correct expression of
the light chain constructs and functional assembly with heavy
chain. Antibodies were detected not only using an anti-human IgG
secondary antibody, but also an anti-rat Ckappa light chain
secondary antibody. The results confirm that the anti-rat Ckappa
antibody (BD Pharmingen #553871, clone MRK-1) recognizes the light
chain expressed by the pSELECT vectors.
[0145] Supernatants were analyzed by non-reducing SDS-PAGE and
Western blot (not shown). Detection using an anti-human IgG heavy
chain antibody did not show bands for supernatants A and B
containing light chain only, as expected. Results for supernatants
C and D were comparable to positive control supernatant E, with a
band close to the 170 kD marker as expected for intact IgG.
Additional lower molecular weight bands were observed as well for
supernatants C, D and E, which might represent degradation
products, IgG fragments resulting from (partial) reduction and/or
irrelevant protein bands due to non-specific binding of the
detection antibody.
[0146] Detection using an anti-rat Ckappa light chain antibody
showed a band close to the 26 kD marker for supernatants A and B,
as expected for light chain only. This band was much more intense
for A compared to B, indicating that the free IGKV1-39 light chain
may be better expressed and/or more stable than the free IGLV2-14
light chain. No bands were detected for control supernatant E as
expected, since the expressed IgG contains a human Ckappa light
chain. For supernatants C and D, expected bands close to the 170 kD
marker were observed; lower molecular weight bands were also
observed, but to a lesser extent than above using the anti-human
IgG antibody.
[0147] In conclusion, transfection of the light chain expression
constructs combined with the heavy chain of anti-tetanus toxoid
(TT) IgG MG1494 resulted in IgG production comparable to the
positive control construct for both the pSELECT kappa and lambda
light chain constructs. Both IgG productions yielded ELISA signals
in a TT ELISA that were better than or comparable to the control
IgG. SDS-PAGE and Western blot analysis confirmed the presence of
intact IgG. The tested anti-rat Ckappa antibody worked efficiently
in both ELISA and Western blot. Culture supernatant from cells
transfected with light chain constructs only did not result in
detectable IgG production nor in detectable TT-specific binding,
while free light chain was detected on Western blot.
Example 8
Expression of IGLV2-14/J-Ck in HEK293 Cell Lines
(pSELECT-IGLV2-14/J-Ck)
[0148] This example describes a method to verify that the
IGLV2-14/J constructs described in Example 6 enable expression and
detection of the IGLV2-14/J-Ck L chain in HEK293 cells. The
IGLV2-14/J-Ck insert (FIG. 7) was modified at the 5' end by
changing the NotI site into a SalI site. This change is required
for cloning of the product into the expression cassette plasmid
pSELECT-hygro (InvivoGen). The CAGGS expression insert
IGLV2-14/J-Ck and pSELECT-hygro were digested with SalI and NheI
ligated and used to transform competentXL1-Blue cells using
standard techniques. Colonies were picked and DNA purified using
Qiagen Midi-prep columns according to the manufacturer's
procedures. The resulting light chain (LC) expressing vector named
0817678_pSELECT.sub.--0815427 was used to transfect HEK293 cells
with Fugene6 (Roche) according to the manufacturer's protocols.
Supernatants were screened for the presence of IGLV2-14/J-Ck light
chains by ELISA and western blot using anti-rat-Ck antibodies
(Becton Dickinson #550336 and 553871) and protocols used in the
art. See Example 7 for details and results.
Example 9
Construction of a VK Promoter-Driven Expression Construct
Containing an IGKV1-39/J Insert and Multiple Enhancer Elements
Derived from the Murine CK Locus (VkP-IGKV1-39/J-Ck; VkP-O12)
[0149] This example describes the construction of an expression
cassette that contains relevant elements to enable B-cell and
developmental/differentiation stage-specific expression of the
rearranged human IGKV1-39 VK region, based on the IGKV1-39 VK
promoter region, leader containing an intron, germline V-gene,
CDR3, IGKJ segment, mouse intergenic region located between Jk and
CK, rat Ck allele a open reading frame, and a mouse intergenic
fragment from the 3' end of the mouse CK gene ending just 3' of the
3' CK enhancer.
[0150] Optimized open reading frames of the leader, IGKV1-39
rearranged gene, and rat CK allele a gene, as described in Example
5, was used for the construction of the expression cassette. The VK
promoter region was obtained by gene synthesis procedures (GeneArt,
GmbH) and is almost identical to the sequence of the human IGKV1-39
region between -500 bp and the ATG (start site) of the gene. The
only deviation from the natural sequence is the introduction of a
GCCACCATGG Kozak sequence (SEQ ID NO:102) at the ATG (start) site
in order to promote translation. A genomic fragment from a mouse
BAC clone (TaconicArtemis) is used as the basis for the
introduction of individual elements. This fragment is identical to
the sequence of the mouse VK locus starting with the intron donor
site located directly 3' of the JK5 region and ending just 3' of
the 3' CK enhancer and covers approximately 12.5 kb.
[0151] The final construct contains from 5' to 3' end the following
elements: human genomic IGKV1-39 promoter (500 bp), a Kozak
sequence, a human IGKV1-39 leader part 1 (optimized), a human
IGKV1-39 leader intron, a human IGKV1-39 leader part 2 (optimized),
a human IGKV1-39 germline gene (optimized), a human J-region
(optimized), a mouse intergenic region including the intron
enhancer element, a rat (Rattus norvegicus) kappa constant region
(optimized), and a mouse intergenic region including the 3' kappa
enhancer. The elements of this expression cassette are shown in
FIG. 8 and named VkP-IGKV1-39/J-Ck (VkP-O12). An outline of the
pVkP-O12 vector and the targeting strategy is depicted in FIGS. 20A
and 21A. The vector was introduced into ES cells following standard
procedures (see Example 14).
Example 10
Construction of a VK Promoter-Driven Expression Construct
Containing an IGLV2-14/J Clone and Multiple CK Locus-Derived
Enhancer Elements (VkP-IGLVL2-14/J-Ck; VkP-2a2)
[0152] This example describes the same construct as described in
Example 9, except that the IGKV1-39 gene and J-region are replaced
by the optimized human IGLV2-14 germline gene including a unique
V-J region (VkP-IGLV2-14/J-Ck; VkP-2a2; FIG. 9).
Example 11
Construction of a VK Promoter-Driven Expression Construct
Containing an IGKV1-39 Clone Lacking the CK Intron Enhancer Element
(VkP-IGKV1-39/J-Ck-.DELTA.1; VkP-O12-del1)
[0153] The construct described in Example 9 was modified by
removing the CK intron enhancer element, located in the intergenic
region between the human J region and the rat CK region by standard
PCR modification and DNA cloning methodologies (GeneArt, GmBH). The
resulting expression cassette is shown in FIG. 10 and named
VkP-IGKV1-39/J-Ck-.DELTA.1 (VkP-O12-del1).
[0154] An outline of the pVkP-O12-del1 vector and the targeting
strategy is depicted in FIGS. 20B and 21B. The vector was
introduced into ES cells following standard procedures (see Example
14).
Example 12
Construction of a VK Promoter-Driven Expression Construct
Containing an IGKV1-39 Clone Lacking the CK Intron Enhancer Element
and a Truncated 3' CK Enhancer Element (VkP-IGKV1-39/J-Ck-.DELTA.2;
VkP-O12-del2)
[0155] The construct described in Example 11 was modified by
truncating the 3' CK enhancer element and deleting part of the
intergenic region 3' of the rat Ck gene, to remove potential
inhibitory elements. This was achieved by removing the intergenic
sequence between an EcoRV site (located 3' of the rat Ck gene) and
the NcoI site present in the 3' enhancer (5993 bp) and further
removing the sequence between the 3' enhancer B stXI site and the B
stXI site 3' of the 3' enhancer (474 bp) using standard methods.
The resulting expression cassette is shown in FIG. 11 and named
VkP-IGKV1-39/J-Ck-.DELTA.2 (VkP-O12-del2).
[0156] An outline of the pVkP-O12-del2 vector and the targeting
strategy is depicted in FIGS. 20C and 21C. The vector was
introduced into ES cells following standard procedures (see Example
14).
Example 13
Expression of Vk Constructs in Cell Lines
[0157] The constructs described in Examples 9-12 are tested for
their ability to produce light chain proteins in the myeloma cell
lines MPC11 (ATCC CCL167), B-cell lymphoma WEHI231 (ATCC CRL-1702),
the T-cell lymphoma EL4 (ATCC TIB-39) and in HEK293 (ATCC CRL1573).
The enhancer and promoter elements in the construct enable
expression in the B-cell lines but not in cell lines derived from
other tissues. After transfection of the cell lines using purified
linearized DNA and Fugene6 (Roche) cells are cultured for transient
expression. Cells and supernatant are harvested and subjected to
SDS-PAGE analysis followed by western blotting using a specific
anti-rat-C-kappa antibody. Supernatants are analyzed in ELISA for
secreted L chains using the anti-rat CK antibody (Beckton Dickinson
#550336).
Example 14
Generation of Transgenic ES Lines
[0158] All constructs as described in Examples 3, 4, 5, 6, 9, 10,
11 and 12 were used to generate individual stable transgenic ES
lines by means of homologous recombination. The methods for
generation of transgenic ES lines via homologous recombination are
known in the field (e.g., Eggan et al., PNAS 98:6209-6214; J.
Seibler, B. Zevnik, B. Kuter-Luks, S. Andreas, H. Kern, T. Hennek,
A. Rode, C. Heimann, N. Faust, G. Kauselmann, M. Schoor, R.
Jaenisch, K. Rajewsky, R. Kuhn, F. Schwenk (2003), Nucleic Acids
Res., February 15; 31(4):e12; Hogan et al. (Cold Spring Harbor
Laboratory Press, Cold Spring Harbor N.Y.), pp. 253-289).
[0159] For all constructs described in Examples 5 and 6, and
Examples 9-12, the RMCE ES cell line (derived from mouse strain
129S6B6F1-Gt(ROSA)26Sortm10Arte) was grown on a mitotically
inactivated feeder layer comprised of mouse embryonic fibroblasts
(MEF) in DMEM High Glucose medium containing 15% FBS (PAN
1302-P220821). Leukemia Inhibitory Factor (Chemicon ESG 1107) was
added to the medium at a concentration of 900 U/mL. For
manipulation, 2.times.10.sup.5 ES-cells were plated on 3.5 cm
dishes in 2 ml medium. Directly before transfection, 2 ml fresh
medium was added to the cells. Three .mu.l Fugene6 Reagent (Roche;
Catalog No. 1 814 443) was mixed with 100 .mu.l serum free medium
(OptiMEM I with Glutamax I; Invitrogen; Catalog No. 51985-035) and
incubated for five minutes. One hundred .mu.l of the Fugene/OptiMEM
solution was added to 2 .mu.g circular vector and 2 .mu.g CAGGS-Flp
and incubated for 20 minutes. This transfection complex was added
dropwise to the cells and mixed. Fresh medium was added to the
cells the following day. From day 2 onwards, the medium was
replaced daily with medium containing 250 .mu.g/mL G418 (Geneticin;
Invitrogen; Catalog No. 10131-019). Seven days after transfection,
single clones were isolated, expanded, and molecular analyzed by
Southern blotting according to standard procedures.
[0160] For each construct, analysis of multiple clones by
restriction enzyme digestion of genomic DNA of single clones
followed by hybridization with 5' probes, 3' probes, and internal
probes resulted in clones that comprised a correct, single
insertion at the correct position in the Rosa26 locus. An example
is provided in FIGS. 14A-C.
Example 15
Generation of Transgenic Mouse Strains
[0161] All ES cell lines that were generated and verified for their
modifications as described in Example 14 were used to generate
stable transgenic mice by means of tetraploid recombination. The
methods are known in the field. In general, after administration of
hormones, superovulated Balb/c females were mated with Balb/c
males. Blastocysts were isolated from the uterus at dpc 3.5. For
microinjection, blastocysts were placed in a drop of DMEM with 15%
FCS under mineral oil. A flat tip, piezo actuated
microinjection-pipette with an internal diameter of 12-15
micrometers was used to inject 10-15 targeted C57BL/6 N.tac ES
cells into each blastocyst. After recovery, injected blastocysts
were transferred to each uterine horn of 2.5 days post coitum,
pseudopregnant NMRI females. Chimerism was measured in chimeras
(G0) by coat color contribution of ES cells to the Balb/c host
(black/white). Highly chimeric mice were bred to strain C57BL/6
females. Depending on the project requirements, the C57BL/6 mating
partners are non-mutant (W) or mutant for the presence of a
recombinase gene (Flp-Deleter or Cre-deleter or CreER inducible
deleter or combination of Flp-deleter/CreER). Germline transmission
was identified by the presence of black, strain C57BL/6, offspring
(G1).
[0162] For example, ESC clone IgVK1-39 2683 8 (see Examples 5 and
14) was injected in a total of 62 blastocysts in three independent
experiments. Three litters were obtained with a total of six pups.
All pups were chimeric. Three heterozygous offspring pups were
obtained that were used for further crossing.
[0163] ESC Clone Kappa 2692 A-C10 (see Examples 3 and 14) was
injected in a total of 54 blastocysts in three independent
experiments. Three litters were obtained with a total of eleven
pups, of which ten were chimeric. Eight heterozygous offspring pups
were obtained that were used for further crossing.
[0164] ESC Clone Kappa 2692 B-C1 (see Examples 3 and 14) was
injected in a total of 51 blastocysts in three independent
experiments. Two litters were obtained with a total of six pups, of
which four were chimeric. Three heterozygous offspring pups were
obtained that were used for further crossing.
Example 16
Breeding
[0165] This example describes the breeding for obtaining mice that
contain transgenic expression cassettes as described Example 14 and
knock-out mice in which the endogenous lambda and kappa loci have
been silenced. The localization of V-lambda on chromosome 16 and
CD19 on chromosome 7 allow standard breeding procedures. The
breeding of the co-localized Vk locus and Rosa26 locus on
chromosome 6 with a distance of about 24 cM requires special
attention during the screening as only a percentage of the
offspring shows crossover in a way that both modifications are
brought together on one chromosome.
[0166] All four loci have to be combined in a single mouse strain
that is homo- or heterozygous for CD19-cre (not described) and
modified Rosa26 transgene and homozygous for the other loci.
Breeding is performed by standard breeding and screening techniques
as appropriate and offered by commercial breeding companies (e.g.,
TaconicArtemis).
Example 17
Immunizations of Mice
[0167] Primary and booster immunization of mice are performed using
standard protocols.
[0168] To validate the transgenic expression of human rearranged
V.kappa. O12 (IGKV1-39)-rat C.kappa. light chains (see Examples 5,
14-16) in B cells from CD19-HuV.kappa.1 mice and to assess its
impact on VH repertoire size, diversity of VH family usage and
V(D)J recombination after immunization, the CD19-HuV.kappa.1
transgenic mice are immunized with tetanus toxin vaccine (TT
vaccine) and VH sequence diversity of randomly picked clones from
CD19-HuV.kappa.1 mice are compared with TT-immunized wt mice and
CD19-Cre HuVk1 negative littermates. Data on the SHM frequency of
the human V.kappa. O12 transgene in the immunized mice are
obtained. A diverse collection of at least 40 TT-specific,
clonally-unrelated mAbs containing the human V.kappa. O12 are
recovered from CD19-HuV.kappa.1 mice by phage display.
[0169] For this, three adult CD19-HuV.kappa.1 mice are vaccinated
with TT vaccine using standard immunization procedures. After
immunization, serum titers are measured using TT specific ELISA
(TT: Statens Serum Institute, Art. no. 2674) and spleen suspensions
subjected to cell sorting by the FACS procedure after staining with
a rat C.kappa.-specific monoclonal antibody to isolate transgenic B
cells (clone RG7/9.1; BD Pharmingen #553901, Lot #06548). RNA from
rat C.kappa.-positive B cells are extracted and the resulting cDNA
material used for library building and SHM analysis.
[0170] The standard monoclonal mouse anti-rat C.kappa. antibody
(clone RG7/9.1; BD Pharmingen #553901, Lot #06548) is used in FACS
analysis of transgene expressing B cells (Meyer et al. (1996), Int.
Immunol. 8:1561). The clone RG7/9.1 antibody reacts with a
monotypic (common) kappa chain determinant. This anti-rat C.kappa.
antibody (clone RG7/9.1 (BD Pharmingen #553901, Lot #06548) is
labeled with R-phycoerythrin (PE) using the LYNX rapid conjugation
kit according to the manufacturer's instructions for FACS analysis
and sorting. The labeled antibody is firstly tested by flow
cytometry for binding to rat CK-containing functional light chain
proteins produced into transiently transfected HEK-293T cells; the
un-conjugated antibody serves as a positive control. Two other
antibodies shown to bind to rat C.kappa. by ELISA and Western-blot
(see Example 7) are tested as well by flow cytometry.
[0171] Fab-phage display library building is carried out with a set
of optimized degenerate PCR primers designed to amplify C57BL/6 VH
genes; the minimal library size is 10.sup.6 clones, and minimal
insert frequency is 80%. The vector used, MV1043 (FIGS. 3 and 12),
contains the human V.kappa. O12 fused to a human C.kappa. region.
The rat C.kappa. is therefore exchanged for the human counterpart
in the library generation process.
[0172] Before selection, VH sequencing of 96 randomly picked clones
is performed to validate VH repertoire diversity that is compared
to diversity obtained from an unselected library previously
generated using the same procedures from BALB/c mice immunized with
TT. A library from C57B1/6 wt mice that are immunized in the same
way allows diversity comparison between two preselected libraries
sharing the same vaccine and the same genetic background.
[0173] Several independent selections are performed on TT coated in
immunotubes. Variables that may be included are selections using
biotinylated antigens in solution or selections on captured TT.
Based on the number and diversity of ELISA-positive clones obtained
in the first selections, decisions on additional rounds of
selection are made. Clones are considered positive when
>3.times. positive over a negative control clone. Positive
clones are analyzed by ELISA against a panel of negative control
antigens to verify antigen specificity. The aim is to identify at
least 40 unique VH regions, as based on unique CDR3 sequences and
V.sub.HDJ.sub.H rearrangements.
[0174] Amplification of the cDNA material from rat
C.kappa.-positive sorted B cells is performed with a PCR forward
primer specific to the human leader sequence and a PCR reverse
primer specific to the rat C.kappa. sequence, in a region not
redundant with the mouse C.kappa. sequence, as reported in a recent
study (Brady et al. (2006), JIM 315:61). Primer combinations and
annealing temperatures are firstly tested on cDNA from HEK-293T
cells transfected with 0817676_pSELECT.sub.--0815426=pSELECT vector
with IGKV1-39 DNA cassette (see Example 7).
[0175] The amplification products is cloned in pJET-1 vector and
after XL1-blue transformation, 96 colonies are sequenced for
assessing VL SHM frequency by direct comparison to the V.kappa. O12
(IGKV1-39) germline sequence. The R/S ratio method, as described in
our study on human TT-specific antibodies (de Kruif et al. (2009),
J. Mol. Biol. 387:548) allows discrimination between random
mutations and antigen-driven mutations that occurred on VL
sequences.
Example 18
Immunofluorescent Analysis of B Cell Populations in Transgenic
Mouse Lines
[0176] This example describes the use of antibodies and flow
cytometry to analyze B cell populations in primary (bone marrow)
and secondary (spleen, peritoneal) lymphoid organs and blood.
Methods and reagents are described in Middendorp et al. (2002), J.
Immunol. 168:2695; and Middendorp et al. (2004), J. Immunol.
172:1371. For analysis of early B cell development in bone marrow,
cells were surface stained with combinations of antibodies (Becton
Dickinson) specific for B220, CD19, CD25, IgM, IgD, mouse Ckappa,
mouse Clambda and rat Ckappa to detect pro-B cells, pre-B cells,
large pre-B cells, early and late immature B cells and
recirculating B cell populations expressing the transgene on their
surface. DAPI staining (Invitrogen) was included to exclude dead
cells from the analysis and FC block (Becton Dickinson) to inhibit
antibody interaction with Fc receptors on myeloid cells. For
analysis of surface transgene expression on B cell populations in
peripheral lymphoid organs and blood, cells were stained with
combinations of antibodies (Becton Dickinson) specific for B220,
CD5, CD19, CD21, CD23, IgM, IgD, mouse Ckappa, mouse Clambda and
rat Ckappa. DAPI staining was included to exclude dead cells from
the analysis and FC block to inhibit antibody interaction with Fc
receptors on myeloid cells. In addition, combinations of antibodies
(Becton Dickinson) specific for CD3, CD4, CD11b, CD11c and NK1.1
were included to determine if transgene expression occurred in cell
types outside of the B cell compartment.
[0177] Three mice heterozygous for the human IGKV1-39/rat Ckappa
transgene and heterozygous for the CD19-Cre transgene on a C57BL6
background (HuVk1/CD19-Cre) were analyzed. As controls for the FACS
analysis, three littermate mice wild-type for the human
IGKV1-39/rat Ckappa transgene and heterozygous for the CD19-Cre
transgene on a C57BL6 background (CD19-Cre) and two C57BL6/NTac
mice (Wt) were included. All animals were allowed to acclimatize in
the animal facility for one week before analysis and all mice were
male and six weeks of age. Lymphocytes were isolated from the
femurs, spleens, peritoneal cavity and blood of mice using
conventional techniques as previously described (Middendorp et al.
(2002), J. Immunol. 168:2695; and Middendorp et al. (2004), J.
Immunol. 172:1371). Antibodies were pre-combined as shown in Table
10 and staining was carried out in 96-well plates. Incubation with
the PE-conjugated anti-rat C kappa (described above) was carried
out before staining with the rat anti-murine antibodies to avoid
non-specific binding. After completion of cell staining, labeled
cells were analyzed on a Becton Dickinson LSR II FACS machine and
the acquired data analyzed with FlowJo software (v6.4.7).
[0178] Transgenic mice were similar in weight, appearance and
activity to wild-type mice. No gross anatomical alterations were
observed during the harvesting of tissues. No difference was
observed in the numbers of B cells in the bone marrow (BM) and
spleen (Table 11) or in the numbers of B cells, T cells and myeloid
cells in peripheral organs between transgenic and wild-type mice.
In addition, the frequency or proportion of the cells in the
different lymphocyte developmental pathways was not altered in
transgenic mice when compared to wild-type mice. Thus in the double
transgenic (HuVk1/CD19-Cre) and transgenic (CD19-Cre) mice lymphoid
and most importantly B cell development was indistinguishable from
wild-type mice.
[0179] In the peripheral lymphoid organs, staining with the
transgene specific antibody (anti-ratCkappa-PE) was only observed
in the B cell populations. T cell, myeloid cell and NK cell
populations were all negative for surface expression of the
transgene in the spleen (FIG. 23). In contrast, in cells stained
with the pan B cell markers B220 and CD19 all cells were shifted to
the right in the FACS plot indicating cell surface expression of
the transgene (FIG. 24). A similar transgene-specific staining was
measured in CD5.sup.+ B1 cells of the peritoneum, a developmentally
distinct population of B cells (FIG. 25).
[0180] Differentiation of B cells from multilineage precursors to
mature B cells occurs in the bone marrow. In the lymphocytes
analyzed from the bone marrow, extracellular and transgene
expression was not detectable in the earliest B cell progenitors
the pro- and pre-B cell consistent with the pattern of normal light
chain expression (FIGS. 26A-B). Transgene expression first becomes
detectable in immature B cells, the developmental stage at which
the germline murine light chain undergoes rearrangement and is
expressed at the cell surface in the context of the preselected
heavy chain (FIGS. 26A-B). Consistent with the staining in the
spleen transgenic light chain expression is also detected on mature
recirculating B cells (FIGS. 26A-B). Thus the CD19-Cre driven
expression of the transgene is consistent with the normal pattern
of light chain expression. The staining with the endogenous light
chain-specific antibody is more intense than that of the
transgene-specific light chain antibody. This may indicate a higher
expression level of the endogenous light chain, a more sensitive
staining with the endogenous light chain-specific antibody or a
combination of both. Importantly, the intensity of the surface
expression of the transgenic light chain is correlated with both
endogenous light chain and IgM surface expression as observed in
staining of circulating B cells in the blood (FIG. 27).
[0181] Thus, overall this analysis demonstrates that expression of
the human IGKV1-39/Ckappa transgene is restricted to the B cell
compartment and the temporal regulation of its expression is
similar to the endogenous kappa and lambda light chains resulting
in normal development of all B cell populations. The apparent lower
level of expression of the transgene could be explained by the
strength of the promoter in comparison to the promoter and
enhancers present on endogenous light chain genes or by a delay in
transgene expression that gives the endogenous light chains a
competitive advantage in pairing with the rearranged heavy chain.
This is consistent with the observation that as B cells mature the
relative intensity of transgene staining increases compared to the
endogenous light chains. In addition, the observation that B cells
numbers are normal and that every surface Ig+ B cell co-expresses
an endogenous and transgenic light chain supports the conclusion
that the IGKV1-39 variable region is capable of pairing with a
normal repertoire of different murine heavy chain variable regions.
We conclude from this analysis that insertion of the IGKV1-39/rat
Ckappa transgene driven by the CD19-Cre activated CAGGS promoter in
the Rosa locus facilitates timely and B cell-specific expression of
the transgene and that the transgene is capable of pairing with a
normal repertoire of murine heavy chains.
Example 19
Epibase.RTM. T-Cell Epitope Profile for IGKV1-39
[0182] The protein sequence of IGKV1-39 (FIG. 12, human germline
IGKV1-39/J Protein) was scanned for the presence of putative HLA
class II restricted epitopes, also known as T.sub.H-epitopes. For
this, Algonomics' Epibase.RTM. platform was applied to IGKV1-39. In
short, the platform analyzes the HLA binding specificities of all
possible 10-mer peptides derived from a target sequence (Desmet et
al. (1992), Nature 356:539-542; Desmet et al. (1997), FASEB J.
11:164-172; Desmet et al. (2002), Proteins 48:31-43; Desmet et al.
(2005), Proteins 58:53-69). Profiling is done at the allotype level
for 20 DRB1, 7 DRB3/4/5, 13 DQ and 7 DP, i.e., 47 HLA class II
receptors in total (see Table 5). Epibase.RTM. calculates a
quantitative estimate of the free energy of binding
.DELTA.G.sub.bind of a peptide for each of the 47 HLA class II
receptors. These data were then further processed as follows:
[0183] Free energies were converted into Kd-values through
.DELTA.G.sub.bind=RT ln(Kd).
[0184] Peptides were classified as strong (S), medium (M), weak and
non (N) binders. The following cutoffs were applied:
[0185] S: strong binder: Kd<0.1 .mu.M.
[0186] M: medium binder: 0.1 .mu.M.ltoreq.Kd<0.8 .mu.M.
[0187] N: weak and non-binder: 0.8 .mu.M.ltoreq.Kd.
[0188] Peptides corresponding to self-peptides were treated
separately. The list of self-peptides was taken from 293 antibody
germline sequences. They are referred to as "germline-filtered"
peptides.
[0189] S- and M-peptides are mapped onto the target sequence in
so-called epitope maps; S-affinities are plotted quantitatively;
M-values are presented qualitatively. As a general overview of the
results, Table 6 lists the number of strong and medium binders in
the analyzed proteins, for the groups of HLA class II receptors
corresponding to the DRB1, DQ, DP and DRB3/4/5 genes. Counting was
done separately for strong and medium affinity binders. Peptides
binding to multiple allotypes of the same group were counted as
one. Values between brackets refer to germline-filtered peptides.
In Table 7, the sequence is shown in a format suitable for
experimental work. The sequence is broken down in consecutive
15-mers overlapping by 12 residues. For each 15-mer, the
promiscuity is listed (the number of allotypes out of a total of 47
for which the 15-mer contains a critical binder), as well as the
implied serotypes. The Epibase.RTM. profile and epitope maps are
shown in FIGS. 16A-C and 17.
[0190] It was concluded that IGKV1-39 contains no strong non-self
DRB1 binders. Typically, significantly more binders were found for
DRB1 than for other HLA genes. This is in agreement with
experimental evidence that allotypes belonging to the DRB1 group
are more potent peptide binders. Medium strength epitopes for DRB1
allotypes are expected to contribute to the population response,
and cannot be disregarded. Again, no non-self DRB1 binders were
found in IGKV1-39.
[0191] In the humoral response raised against an antigen, the
observed T.sub.H cell activation/proliferation is generally
interpreted in terms of the DRB1 specificity. However, one cannot
ignore the possible contribution of the DRB3/4/5, DQ and DP genes.
Given the lower expression levels of these genes as compared to
DRB1, the focus was on the class of strong epitopes for DRB3/4/5,
DQ and DP. "Critical epitopes" are those epitopes that are strong
binders for any DRB1, DRB3/4/5, DQ or DP allotype or are medium
binders for DRB1. IGKV1-39 contains no strong or medium non-self
binders for DRB3/4/5, DQ, or DP.
[0192] A number of peptides are also present in germline sequences
(values between brackets in Table 6). Such peptides may very well
bind to HLA but they are assumed to be self and, hence,
non-immunogenic. In total, six strong and 16 medium
germline-filtered DRB1 binders were found in IGKV1-39. Framework
region 1 up to framework region 3 is an exact match for germline
V-segment VKI 2-1-(1) O12 (VBase), a.k.a. IGKV1-39*01 (IMGT).
Framework region 4 is an exact match for germline J-segment JK1
(V-base) a.k.a. IGKJ1*01(IMGT). It is hardly surprising that these
segments do not contain any non-self epitopes.
Example 20
Production Characteristics of IGKV1-39
[0193] There is a great demand for antibody discovery platforms
that yield therapeutic antibodies that are thermodynamically stable
and give good expression yields. These characteristics are
important in ensuring the stability of the drug substance during
production and after injection of the drug product into the
patient. In addition good expression yields impact directly on the
cost of drug manufacture and thus pricing, patient access and
profitability. Virtually all therapeutic antibodies in clinical use
today are composed of human IgG1 and kappa constant regions but use
different heavy and light chain variable regions that confer
specificity. Human variable heavy and light chain domains can be
divided into families that have greater than 80% sequence
divergence. When rearranged examples of these families in germline
configuration are combined and compared for stability and yield it
is clear that the gene families are not equal in terms of
biophysical properties. In particular V.sub.H3, V.sub.H1 and
V.sub.H5 have favourable stability for the heavy chains and Vk1 and
Vk3 have the best stability and yield of light chains. In addition
when mutations are introduced as part of the somatic hypermutation
process they can interfere with V.sub.H/V.sub.L pairing. To assess
the effect that different light chain genes with different rates of
mutation have on the production characteristics of a fixed V.sub.H
chain, a Fab phage display library was built of light chains (kappa
and lambda) from six naive healthy donors combined with a panel of
44 TT binding heavy chains from immunized donors. After one round
of selection TT binding Fab clones were isolated. Several of these
shared the same V.sub.H gene as the TT clone PG1433 in combination
with different light chains. The Fab light chain fragments were
recloned into a kappa expression vector and transfected in
combination with DNA encoding the heavy chain of PG1433 into 293
cells and specific IgG production measured by ELISA. As
demonstrated in Table 8 the selected clones containing PG1433
V.sub.H combined with different light chains had between five- and
ten-fold lower protein expression PG1433 V.sub.H combined with
IGKV1-39. Note that all of the light chains contained amino acid
mutations within their coding regions that might disrupt V.sub.H
paring and reduce production stability. Thus, in addition to
reducing the chances of unwanted immunogenicity, it is expected
that the use of the light chain IGKV1-39 without mutations
contributes to improved production stability and yields of various
specificity-contributing V.sub.H genes. Indeed stable clones
generated by the transfection of different V.sub.H genes all paired
with IGKV1-39 are able to be passaged extensively and still retain
robust production characteristics as shown in Table 9.
Example 21
Generation of Mice Expressing Fully Human VH and VL Regions
[0194] Transgenic mice described herein are crossed with mice that
already contain a human VH locus. Examples of appropriate mice
comprising a human VH locus are disclosed in Taylor et al. (1992),
Nucleic Acids Res. 20:6287-95; Lonberg et al. (1994), Nature
368:856-9; Green et al. (1994), Nat. Genet. 7:13-21; Dechiara et
al. (2009), Methods Mol. Biol. 530:311-24.).
[0195] After crossing and selecting for mice that are at least
heterozygous for the IGKV1-39 transgene and the human VH locus,
selected mice are immunized with a target. VH genes are harvested
as described hereinabove. This method has the advantage that the VH
genes are already fully human and thus do not require
humanization.
Example 22
Isolation, Characterization, Oligoclonics Formatting and Production
of Antibodies Targeting Human IL6 for Treatment of Chronic
Inflammatory Diseases Such as Rheumatoid Arthritis
[0196] A spleen VH repertoire from transgenic mice that are
immunized with human recombinant IL6 is cloned in a phage display
Fab vector with a single human IGKV1-39-C kappa light chain
(identical to the mouse transgene) and subjected to panning against
the immunogen human IL6. Clones that are obtained after two to four
rounds of panning are analyzed for their binding specificity. VH
genes encoding IL6-specific Fab fragments are subjected to sequence
analysis to identify unique clones and assign VH, DH and JH
utilization. The Fab fragments are reformatted as IgG1 molecules
and transiently expressed. Unique clones are then grouped based on
non-competition in binding assays and subjected to affinity and
functional analysis. The most potent anti-IL6 IgG1 mAbs are
subsequently expressed as combinations of two, three, four or five
heavy chains comprising different VH-regions in the Oligoclonics
format, together with one IGKV1-39-C-based kappa light chain and
tested in vitro for complex formation with IL-6. The Oligoclonics
are also tested in vivo for clearance of human IL-6 from mice. An
Oligoclonic with the most potent clearance activity is chosen and
the murine VH genes humanized according to conventional methods.
The humanized IgG1 are transfected into a mammalian cell line to
generate a stable clone. An optimal subclone is selected for the
generation of a master cell bank and the generation of clinical
trial material.
[0197] Many of the protocols described here are standard protocols
for the construction of phage display libraries and the panning of
phages for binding to an antigen of interest and are described, for
example, in Antibody Phage Display: Methods and Protocols (2002),
Editor(s) Philippa M. O'Brien, Robert Aitken, Humana Press, Totowa,
N.J., USA.
Immunizations
[0198] Transgenic mice receive three immunizations with human IL6
every two weeks using the adjuvant Sigma titerMax according to
manufacturer's instructions.
RNA Isolation and cDNA Synthesis
[0199] Three days after the last immunization, spleens and
lymphnodes from the mice are removed and passed through a 70 micron
filter into a tube containing PBS pH 7.4 to generate a single cell
suspension. After washing and pelleting of lymphocytes, cells are
suspended in TRIzol LS Reagent (Invitrogen) for the isolation of
total RNA according to the manufacturer's protocol and subjected to
reverse transcription reaction using 1 microgram of RNA,
Superscript III RT in combination with dT20 according to
manufacturer's procedures (Invitrogen).
[0200] The generation of Fab phage display libraries is carried out
as described in Example 2.
Selection of Phages on Coated Immunotubes
[0201] Human recombinant IL6 is dissolved in PBS in a concentration
of 5 .mu.g/ml and coated to MAXISORP.TM. Nunc-Immuno Tube (Nunc
444474) overnight at 4.degree. C. After discarding the coating
solution, the tubes are blocked with 2% skim milk (ELK) in PBS
(blocking buffer) for one hour at Room Temperature (RT). In
parallel, 0.5 ml of the phage library is mixed with 1 ml blocking
buffer and incubated for 20 minutes at room temperature. After
blocking the phages, the phage solution is added to the IL6-coated
tubes and incubated for two hours at RT on a slowly rotating
platform to allow binding. Next, the tubes are washed ten times
with PBS/0.05% TWEEN.TM.-20 detergent followed by phage elution by
incubating with 1 ml 50 mM glycine-HCl pH 2.2 ten minutes at RT on
rotating wheel and directly followed by neutralization of the
harvested eluent with 0.5 ml 1 M Tris-HCl pH 7.5.
Harvesting Phage Clones
[0202] A 5 ml XL1-Blue MRF (Stratagene) culture at O.D. 0.4 is
added to the harvested phage solution and incubated for 30 minutes
at 37.degree. C. without shaking to allow infection of the phages.
Bacteria are plated on Carbenicillin/Tetracycline 4% glucose 2*TY
plates and grown overnight at 37.degree. C.
Phage Production
[0203] Phages are grown and processed as described by Kramer et al.
2003 (Kramer et al. 2003, Nucleic Acids Res. 31(11):e59) using
VCSM13 as helper phage strain.
Phage ELISA
[0204] ELISA plates are coated with 100 microliters human
recombinant IL6 per well at a concentration of 2.5 micrograms/ml in
PBS overnight at 4.degree. C. Plates coated with 100 microliters
thyroglobulin at a concentration of 2 micrograms/ml in PBS are used
as a negative control. Wells are emptied, dried by tapping on a
paper towel, filled completely with PBS-4% skimmed milk (ELK) and
incubated for one hour at room temperature to block the wells.
After discarding the block solution, phage minipreps pre-mixed with
50 .mu.l blocking solution are added and incubated for one hour at
RT. Unbound phages are subsequently removed by five washing steps
with PBS-0.05% Tween-20. Bound phages are detected by incubating
the wells with 100 microliters anti-M13-HRP antibody conjugate
(diluted 1/5000 in blocking buffer) for one hour at room
temperature. Free antibody is removed by repeating the washing
steps as described above, followed by TMB substrate incubation
until color development was visible. The reaction is stopped by
adding 100 microliters of 2 M H2504 per well and analyzed on an
ELISA reader at 450 nm emission wavelength.
Sequencing
[0205] Clones that give signals at least three times above the
background signal are propagated, used for DNA miniprep procedures
(see procedures Qiagen miniPrep manual) and subjected to nucleotide
sequence analysis. Sequencing is performed according to the Big Dye
1.1 kit accompanying manual (Applied Biosystems) using a reverse
primer (CH1_Rev1, Table 1) recognizing a 5' sequence of the CH1
region of the human IgG1 heavy chain (present in the Fab display
vector MV1043, FIGS. 3 and 12). The sequences of the murine VH
regions are analyzed for diversity of DH and JH gene segments.
Construction and Expression of Chimeric IgG1
[0206] Vector MV1057 (FIGS. 12 and 22) was generated by cloning the
transgene (IGKV1-39) L chain fragment into a derivative of vector
pcDNA3000Neo (Crucell, Leiden, The Netherlands) that contains the
human IgG1- and kappa constant regions. VH regions are cloned into
MV 1057 and nucleotide sequences for all constructs are verified
according to standard techniques. The resulting constructs are
transiently expressed in HEK293T cells and supernatants containing
chimeric IgG1 are obtained and purified using standard procedures
as described before (M. Throsby 2006, J. Virol. 80:6982-92).
IgG1 Binding and Competition Analysis
[0207] IgG1 antibodies are titrated in ELISA using IL6-coated
plates as described above and an anti-human IgG peroxidase
conjugate. Competition ELISAs to group antibodies based on epitope
recognition are performed by incubating Fab phages together with
IgG1 or with commercial antibodies against IL6 (e.g., Abcam cat.
no. ab9324) in IL6-coated plates, followed by detection of bound
Fab phage using an anti-M13 peroxidase conjugate.
IgG 1 Affinity Measurements
[0208] The affinities of the antibodies to IL6 are determined with
the Quantitative kinetic protocol on the Octet (ForteBio).
Antibodies are captured onto an Anti-Human IgG Fc Capture biosensor
and exposed to free IL6 and analyzed using proprietary software to
calculate the Kd of each antibody.
Functional Activity of IL6 Antibodies
[0209] To test the ability of the selected antibodies to inhibit
binding between IL6 and IL6 receptor (IL6R), an ELISA based assay
is used. Various concentrations of antibody are mixed with a fixed
concentration (10 ng/ml) of biotinylated IL6 as described by Naoko
et al. 2007, Can. Res. 67:817-875. The IL6-antibody immune complex
is added to immobilized IL6R. The binding of biotinylated IL6 to
IL6R is detected with horseradish peroxidase-conjugated
streptavidin. The reduction of ELISA signal is a measurement of
inhibition. As positive control for inhibition of binding between
IL6 and IL6R either anti-IL6R antibody (Abcam cat. no. ab34351;
clone B-R6) or anti IL6 antibody (Abcam cat. no. ab9324) is
used.
[0210] In vitro blocking activity of the selected anti-IL6
antibodies is measured in a proliferation assay using the
IL6-dependent cell line 7TD1. Briefly, cells are incubated with
different concentrations of human IL6 with or without the anti-IL6
antibody. The available amount of IL6 determines the degree of
proliferation. Thus if an added antibody blocks IL6 binding the
proliferation readout is reduced compared to a non binding antibody
control. Proliferation is measured by the incorporation of
5-bromo-2'-deoxy-uridine (BrdU) into the DNA using the BrdU
proliferation kit (Roche cat. no. 11444611001) according to the
manufacturer's instructions.
Generation of Anti-IL6 Oligoclonics
[0211] The most potent anti-IL6 antibodies are selected from each
epitope group. The expression constructs expressing these
antibodies are transfected into HEK293T cells in non-competing
groups of three in different ratios (1:1:1; 3:1:1; 1:3:1; 1:1:3;
3:3:1; 1:3:3; 3:1:3; 10:1:1; 1:10:1; 1:1:10; 10:10:1; 1:10:10;
10:1:10; 3:10:1; 10:3:1; 1:10:3; 3:1:10; 10:1:3; 1:3:10). Antibody
containing supernatants are harvested and purified and
characterized as above.
Complex Formation and In Vivo Clearance of Anti-IL6
Oligoclonics
[0212] To measure the ability of anti-IL6 Oligoclonics to form
immune complexes and to analyze these complexes Size Exclusion
Chromatography (SEC) is used according to the approach disclosed by
Min-Soo Kim et al. (2007), JMB 374:1374-1388, to characterize the
immune-complexes formed with different antibodies to TNF.alpha..
Different molar ratios of the anti-IL6 Oligoclonics are mixed with
human IL6 and incubated for 20 hours at 4.degree. C. or 25.degree.
C. The mixture is analyzed on an HPLC system fitted with a size
exclusion column; different elution times are correlated to
molecular weight using a molecular weight standards.
[0213] The ability of antibodies to form complexes with IL6 is
correlated with their ability to rapidly clear the cytokine from
the circulation in vivo. This is confirmed by measuring the
clearance of radiolabelled IL6 from mice. Briefly, female, six- to
eight-week-old Balb/c mice are obtained and 18 hours before the
experiment, the animals are injected intravenously (IV) via the
lateral tail vein with different doses of purified anti-IL6
Oligoclonics. On day 0, the mice are injected IV with 50
microliters of radiolabeled IL-6 (1.times.10E7 cpm/mL) under the
same conditions. Blood samples (approximately 50 microliters) are
collected at several time intervals and stored at 4.degree. C. The
samples are centrifuged for five minutes at 4000.times.g and the
radioactivity of the serum determined. All pharmacokinetic
experiments are performed simultaneously with three animals for
each treatment.
Generation of Anti-IL6 Oligoclonics Stable Clones and Preclinical
Development
[0214] A lead anti-IL6 Oligoclonic is selected based on the in
vitro and in vivo potency as determined above. The murine VH genes
are humanized according to standard methods and combined with the
fully human IGKV1-39 light chain in an expression vector as
described above. Examples of humanization methods include those
based on paradigms such as resurfacing (E. A. Padlan et al. (1991),
Mol. Immunol. 28:489), superhumanization (P. Tan, D. A., et al.
(2002), J. Immunol. 169:1119) and human string content optimization
(G. A. Lazar et al. (2007), Mol. Immunol. 44:1986). The three
constructs are transfected into PER.C6 cells at the predetermined
optimal ratio (described above) under the selective pressure of
G418 according to standard methods. A stable high producing
anti-IL6 Oligoclonic clone is selected and a working and qualified
master cell bank generated.
TABLE-US-00002 TABLE 1 List of primers DO- Primer Sequence 0012
CH1_Rev1 TGCCAGGGGGAAGACCGATG (SEQ ID NO: 4) 0656 MVH-1
GCCGGCCATGGCCGAGGTRMAGCTTCAGGAGTCAGGAC (SEQ ID NO: 5) 0657 MVH-2
GCCGGCCATGGCCGAGGTSCAGCTKCAGCAGTCAGGAC (SEQ ID NO: 6) 0658 MVH-3
GCCGGCCATGGCCCAGGTGCAGCTGAAGSASTCAGG (SEQ ID NO: 7) 0659 MVH-4
GCCGGCCATGGCCGAGGTGCAGCTTCAGGAGTCSGGAC (SEQ ID NO: 8) 0660 MVH-5
GCCGGCCATGGCCGARGTCCAGCTGCAACAGTCYGGAC (SEQ ID NO: 9) 0661 MVH-6
GCCGGCCATGGCCCAGGTCCAGCTKCAGCAATCTGG (SEQ ID NO: 10) 0662 MVH-7
GCCGGCCATGGCCCAGSTBCAGCTGCAGCAGTCTGG (SEQ ID NO: 11) 0663 MVH-8
GCCGGCCATGGCCCAGGTYCAGCTGCAGCAGTCTGGRC (SEQ ID NO: 12) 0664 MVH-9
GCCGGCCATGGCCCAGGTYCAGCTYCAGCAGTCTGG (SEQ ID NO: 13) 0665 MVH-10
GCCGGCCATGGCCGAGGTCCARCTGCAACAATCTGGACC (SEQ ID NO: 14) 0666 MVH-11
GCCGGCCATGGCCCAGGTCCACGTGAAGCAGTCTGGG (SEQ ID NO: 15) 0667 MVH-12
GCCGGCCATGGCCGAGGTGAASSTGGTGGAATCTG (SEQ ID NO: 16) 0668 MVH-13
GCCGGCCATGGCCGAVGTGAAGYTGGTGGAGTCTG (SEQ ID NO: 17) 0669 MVH-14
GCCGGCCATGGCCGAGGTGCAGSKGGTGGAGTCTGGGG (SEQ ID NO: 18) 0670 MVH-15
GCCGGCCATGGCCGAKGTGCAMCTGGTGGAGTCTGGG (SEQ ID NO: 19) 0671 MVH-16
GCCGGCCATGGCCGAGGTGAAGCTGATGGARTCTGG (SEQ ID NO: 20) 0672 MVH-17
GCCGGCCATGGCCGAGGTGCARCTTGTTGAGTCTGGTG (SEQ ID NO: 21) 0673 MVH-18
GCCGGCCATGGCCGARGTRAAGCTTCTCGAGTCTGGA (SEQ ID NO: 22) 0674 MVH-19
GCCGGCCATGGCCGAAGTGAARSTTGAGGAGTCTGG (SEQ ID NO: 23) 0675 MVH-20
GCCGGCCATGGCCGAAGTGATGCTGGTGGAGTCTGGG (SEQ ID NO: 24) 0676 MVH-21
GCCGGCCATGGCCCAGGTTACTCTRAAAGWGTSTGGCC (SEQ ID NO: 25) 0677 MVH-22
GCCGGCCATGGCCCAGGTCCAACTVCAGCARCCTGG (SEQ ID NO: 26) 0678 MVH-23
GCCGGCCATGGCCCAGGTYCARCTGCAGCAGTCTG (SEQ ID NO: 27) 0679 MVH-24
GCCGGCCATGGCCGATGTGAACTTGGAAGTGTCTGG (SEQ ID NO: 28) 0680 MVH-25
GCCGGCCATGGCCGAGGTGAAGGTCATCGAGTCTGG (SEQ ID NO: 29) 0681 ExtMVH-1
CAGTCACAGATCCTCGCGAATTGGCCCA ATGGCCSANG (SEQ ID NO: 30) 0682
ExtMVH-2 CAGTCACAGATCCTCGCGAATTGGCCCA ATGGCCSANC (SEQ ID NO: 31)
0683 MJH-Rev1 GGGGGTGTCGTTTTGGCTGAGGAGAC GTGG (SEQ ID NO: 32) 0684
MJH-Rev2 GGGGGTGTCGTTTTGGCTGAGGAGAC GTGG (SEQ ID NO: 33) 0685
MJH-Rev3 GGGGGTGTCGTTTTGGCTGCAGAGAC AGAG (SEQ ID NO: 34) 0686
MJH-Rev4 GGGGGTGTCGTTTTGGCTGAGGAGAC GAGG (SEQ ID NO: 35) 0687
ExtMJH-Rev1& GGGGGTGTCGTTTTGGCTGAGGAGAC GTGG (SEQ ID NO: 36)
0688 ExtMJH-Rev2in GGGGGTGTCGTTTTGGCTGAGGAGAC GTGG (SEQ ID NO: 37)
0690 ExtMJH-Rev3 GGGGGTGTCGTTTTGGCTGAGGAGAC AGAG (SEQ ID NO: 38)
0691 ExtMJH-Rev4 GGGGGTGTCGTTTTGGCTGAGGAGAC GAGG (SEQ ID NO:
39)
TABLE-US-00003 TABLE 2 Phage ELISA signal levels as measured at 450
nm. TT-coated plates represent plates that were coated with tetanus
toxoid. Thyroglobulin-coated plates are used as negative controls.
10/10 and 15/15 indicate the number of wash steps with PBS-Tween
during panning procedures. The 10/10 tetanus toxoid and 10/10
thyroglobulin plates and the 15/15 tetanus toxoid and 15/15
thyroglobulin plates are duplicates from each other except for the
coating agent. OD values higher than three times the background are
assumed specific. 1 2 3 4 5 6 7 8 9 10 11 12 TT-coated plate 10/10
washings A 0.139 0.093 0.089 0.121 0.117 0.598 0.146 0.115 0.18
0.155 0.543 0.601 B 0.136 0.404 0.159 0.187 0.489 0.134 0.216 0.092
0.222 0.108 0.181 0.484 C 0.197 0.526 0.09 0.213 0.395 0.155 0.108
0.12 0.183 0.136 0.092 0.866 D 0.143 0.258 0.101 0.422 0.088 0.243
0.485 0.251 0.304 0.198 0.478 0.091 E 0.445 0.169 0.526 0.481 0.206
0.285 0.111 0.119 0.128 0.2 0.118 0.098 F 0.237 0.291 0.594 0.139
0.206 0.565 0.543 0.091 0.136 0.227 0.228 0.099 G 0.459 0.102 0.152
0.659 0.203 0.452 0.152 0.133 0.094 0.102 0.375 0.098 H 0.341 0.623
0.745 0.415 0.682 0.527 0.655 0.114 0.258 0.284 0.685 0.113
TT-coated plate 15/15 washings A 0.247 0.582 0.421 0.428 0.133
0.082 0.262 0.079 0.343 0.414 0.095 0.292 B 0.065 0.364 0.073 0.042
0.049 0.071 0.046 0.103 0.078 0.057 0.048 0.155 C 0.081 0.044 0.066
0.082 0.225 0.444 0.203 0.362 0.122 0.047 0.052 0.309 D 0.092 0.11
0.59 0.22 0.33 0.544 0.058 0.159 0.047 0.174 0.086 0.05 E 0.469
0.577 0.206 0.304 0.13 0.749 0.431 0.062 0.167 0.049 0.056 0.049 F
0.846 0.07 0.561 0.656 0.882 0.094 0.383 0.13 0.152 0.098 0.134
0.048 G 0.537 0.052 0.49 0.105 0.337 0.193 0.514 0.294 0.068 0.35
0.525 0.05 H 0.061 0.306 0.157 0.853 0.054 0.534 0.102 0.235 0.441
0.412 0.565 0.061 Thyroglobulin-coated plate 10/10 washings A 0.047
0.051 0.045 0.043 0.051 0.044 0.046 0.042 0.047 0.048 0.049 0.05 B
0.042 0.042 0.042 0.042 0.043 0.041 0.041 0.042 0.043 0.045 0.042
0.046 C 0.044 0.043 0.043 0.044 0.043 0.044 0.043 0.042 0.043 0.041
0.044 0.046 D 0.045 0.044 0.044 0.044 0.045 0.046 0.045 0.056 0.045
0.049 0.048 0.73 E 0.046 0.045 0.046 0.044 0.045 0.044 0.044 0.044
0.047 0.046 0.047 0.926 F 0.048 0.045 0.044 0.046 0.044 0.043 0.044
0.046 0.046 0.046 0.046 0.792 G 0.051 0.048 0.045 0.045 0.044 0.043
0.048 0.045 0.048 0.051 0.045 0.053 H 0.064 0.05 0.049 0.047 0.05
0.051 0.047 0.046 0.047 0.047 0.047 0.056 Thyroglobulin-coated
plate 15/15 washings A 0.036 0.049 0.045 0.044 0.046 0.047 0.046
0.042 0.042 0.043 0.042 0.041 B 0.045 0.042 0.041 0.043 0.043 0.043
0.045 0.045 0.047 0.048 0.044 0.045 C 0.049 0.047 0.047 0.046 0.046
0.046 0.045 0.047 0.046 0.045 0.045 0.052 D 0.047 0.049 0.048 0.048
0.048 0.048 0.047 0.052 0.048 0.046 0.048 0.456 E 0.049 0.047 0.047
0.047 0.047 0.049 0.047 0.048 0.047 0.046 0.048 0.412 F 0.05 0.047
0.046 0.046 0.046 0.046 0.046 0.046 0.046 0.047 0.048 0.528 G 0.05
0.048 0.045 0.045 0.046 0.049 0.048 0.046 0.053 0.049 0.05 0.057 H
0.057 0.05 0.046 0.045 0.047 0.049 0.047 0.047 0.046 0.047 0.053
0.048
TABLE-US-00004 TABLE 3 Protein sequence analysis of ELISA positive
tetanus toxoid binders. CDR3 sequence, CDR3 length, VH family
members and specific name, JH origin and DH origin of the clones is
indicated. CDR3/SEQ ID NO: CDR3 length VH DH JH V Gene family
HGAYYTYDEKAWFAY (SEQ ID NO: 40) 15 musIGHVl92 DSP2.11 JH3 mouse
VH7183 HGAYYTYDEKAWFAY (SEQ ID NO: 40) 15 musIGHVl92 DSP2.11 JH3
mouse VH7183 HGAYYTYDEKAWFAY (SEQ ID NO: 40) 15 musIGHVl92 DSP2.11
JH3 mouse VH7183 HGAYYTYDEKAWFAY (SEQ ID NO: 40) 15 musIGHVl92
DSP2.11 JH3 mouse VH7183 HGAYYTYDEKAWFAY (SEQ ID NO: 40) 15
musIGHVl92 DSP2.11 JH3 mouse VH7183 HGAYYTYDEKAWFAY (SEQ ID NO: 40)
15 musIGHVl92 DSP2.11 JH3 mouse VH7183 HGAYYTYDEKAWFAY (SEQ ID NO:
40) 15 musIGHVl92 DSP2.11 JH3 mouse VH7183 HGAYYTYDEKAWFAY (SEQ ID
NO: 40) 15 musIGHVl92 DSP2.11 JH3 mouse VH7183 HGAYYTYDEKAWFAY (SEQ
ID NO: 40) 15 musIGHVl92 DSP2.11 JH3 mouse VH7183 HGAFYTYDEKPWFAY
(SEQ ID NO: 41) 15 musIGHVl92 IGHD2-14*01 JH3 mouse VH7183
HISYYRYDEEVSFAY (SEQ ID NO: 42) 15 musIGHVl92 IGHD2-14*01 JH3 mouse
VH7183 HISYYRYDEEVSFAY (SEQ ID NO: 42) 15 musIGHVl92 IGHD2-14*01
JH3 mouse VH7183 GWRAFAY (SEQ ID NO: 43) 7 musIGHVl3l DSP2.9 JH3
mouse VH7183 GWRAFAY (SEQ ID NO: 43) 7 musIGHVl3l DSP2.9 JH3 mouse
VH7183 GWRAFAY (SEQ ID NO: 43) 7 musIGHVl3l DSP2.9 JH3 mouse VH7183
DRGNYYGMDY (SEQ ID NO: 44) 10 musIGHVl78 DSP2.1 JH4 mouse VH7183
LGDYYVDWFFAV (SEQ ID NO: 45) 12 musIGHVl65 DFL16.1 JH1 mouse VH7183
NFPAWFAF (SEQ ID NO: 46) 8 musIGHV547 DST4.3inv JH3 mouse VJH558
NFPAWFAY (SEQ ID NO: 46) 8 musIGHV547 DSP2.1 JH3 mouse VJH558
NFPAWFVY (SEQ ID NO: 46) 8 musIGHV547 DSP2.1 JH3 mouse VJH558
SFTPVPFYYGYDWYFDV (SEQ ID NO: 47) 17 musIGHV532 DSP2.3 JH1 mouse
VJH558 SFTPVPFYYGYDWYFDV (SEQ ID NO: 47) 17 musIGHV532 DSP2.3 JH1
mouse VJH558 SDYDWYFDV (SEQ ID NO: 48) 9 musIGHV286 DSP2.2 JH1
mouse VJH558 SDYDWYFDV (SEQ ID NO: 48) 9 musIGHV286 DSP2.2 JH1
mouse VJH558 DSKWAYYFDY (SEQ ID NO: 49) 10 musIGHV532 DST4.3 JH2
mouse VJH558 GDYTGYGMDY (SEQ ID NO: 50) 10 musIGHV125 DSP2.13 JH4
mouse VHSM7 GDYTGYGMDY (SEQ ID NO: 50) 10 musIGHV125 DSP2.13 JH4
mouse VHSM7 GGYDGYWFPY (SEQ ID NO: 51) 10 musIGHVl25 DSP2.9 JH3
mouse VHSM7
TABLE-US-00005 TABLE 4 Vector combinations that were transfected to
HEK293T. Combined Conc. Code HC vector LC vector vector Prep name
(.mu.g/ml) A x 0817676_pSELECT_0815426 x PIGKV1-39/ -- (IGKV1-39)
P1 B x 0817678_pSELECT_0815427 x PIGLV2-14/ -- (IGLV2-14) P1 C
MV1110 0817676_pSELECT_0815426 x PMV1110/ 11.0 (IGKV1-39)
IGKV1-39/P1 D MV1110 0817678_pSELECT_0815427 x PMV1110/ 15.4
(IGLV2-14) IGLV2-14/P1 E x x MG1494 MG1494/P2 16.1
TABLE-US-00006 TABLE 5 HLA allotypes considered in T.sub.H-epitope
profiling. The corresponding serotypes are shown, as well as
allotype frequencies in the Caucasian population (Klitz et al.
(2003), Tissue Antigens 62: 296-307; Gjertson and Terasake (eds)
in: HLA 1997; Gjertson and Terasake (eds) in: HLA 1998; Castelli et
al. (2002), J. Immunol. 169: 6928-6934). Frequencies can add up to
more than 100% since each individual has two alleles for each gene.
If all allele frequencies of a single gene were known, they would
add up to slightly less than 200% due to homozygous individuals.
HLA type Serotype Population % DRB1*0101 DR1 17.4 DRB1*0102 DR1 4.9
DRB1*0301 DR17(3) 21.2 DRB1*0401 DR4 11.5 DRB1*0402 DR4 3.1
DRB1*0404 DR4 5.5 DRB1*0405 DR4 2.2 DRB1*0407 DR4 <2 DRB1*0701
DR7 23.4 DRB1*0801 DR8 3.3 DRB1*0802 DR8 <2 DRB1*0901 DR9 <2
DRB1*1101 DR11(5) 17 DRB1*1104 DR11(5) 5.7 DRB1*1201 DR12(5) 3.1
DRB1*1301 DR13(6) 15.4 DRB1*1302 DR13(6) 10.8 DRB1*1401 DR14(6) 4.2
DRB1*1501 DR15(2) 13.2 DRB1*1601 DR16(2) 5.5 DRB1*0101 DR52 24.6
DRB1*0202 DR52 43 DRB1*0301 DR52 10 DRB1*0101 DR53 25.5 DRB4*0103
DR53 21 DRB5*0101 DR51 15.8 DRB5*0202 DR51 5.7 DQA1*0101/DQB1*0501
DQ5(1) 20.5 DQA1*0102/DQB1*0502 DQ5(1) 2.6 DQA1*0102/DQB1*0602
DQ6(1) 26.5 DQA1*0102/DQB1*0604 DQ6(1) 6.7 DQA1*0103/DQB1*0603
DQ6(1) 11 DQA1*0104/DQB1*0503 DQ5(1) 4 DQA1*0201/DQB1*0202 DQ2 20.9
DQA1*0201/DQB1*0303 DQ9(3) 7.2 DQA1*0301/DQB1*0301 DQ7(3) 12.5
DQA1*0301/DQB1*0302 DQ8(3) 18.3 DQA1*0401/DQB1*0402 DQ4 4.5
DQA1*0501/DQB1*0201 DQ2 24.6 DQA1*0501/DQB1*0301 DQ7(3) 20.9
DPA1*0103/DPB1*0201 DPw2 19.9 DPA1*0103/DPB1*0401 DPw4 65.1
DPA1*0103/DPB1*0402 DPw4 24.3 DPA1*0201/DPB1*0101 DPw1 6.3
DPA1*0201/DPB1*0301 DPw3 <2 DPA1*0201/DPB1*0501 DPw5 <2
DPA1*0201/DPB1*0901 -- 2.4
TABLE-US-00007 TABLE 6 T.sub.H epitope counts for IGKV1-39.
Peptides binding to multiple HLAs of the same group (DRB1,
DRB3/4/5, DP, DQ) are counted as one. Values between brackets refer
to germline-filtered peptides. DRB1 DRB3/4/5 DQ DP Strong Medium
Strong Medium Strong Medium Strong Medium Merus IGKV1-39 0 (+6) 0
(+16) 0 (+0) 0 (+5) 0 (+3) 0 (+9) 0 (+0) 0 (+9)
TABLE-US-00008 TABLE 7 Mapping of Epibase .RTM. predictions for
Merus IGKV1-39 in the classical 15-mer peptide format. This table
shows the allotype count of critical epitopes (SEQ ID NOs: 52-83)
and implicated serotypes for each of the 15-mers spanning the Merus
IGKV1-39 sequence. Start Allotype 15mer position 15-mer sequence
count Implicated serotypes 1 1 DIQMTQSPSSLSASV 6 DR1, DR4, DR7, DR9
2 4 MTQSPSSLSASVGDR 5 DR1, DR4, DR9 3 7 SPSSLSASVGDRVTI 0 4 10
SLSASVGDRVTITCR 0 5 13 ASVGDRVTITCRASQ 0 6 16 GDRVTITCRASQSIS 2
DR11(5), DR7 7 19 VTITCRASQSISSYL 4 DQ2, DR11(5), DR4, DR7 8 22
TCRASQSISSYLNWY 2 DQ2, DR4 9 25 ASQSISSYLNWYQQK 5 DR13(6), DR15(2),
10 28 SISSYLNWYQQKPGK 8 DR12(5), DR13(6), DR15(2), DR16(2), DR4,
DR8 11 31 SYLNWYQQKPGKAPK 10 DR1, DR12(5), DR16(2), DR4, DR51, DR8
12 34 NWYQQKPGKAPKLLI 9 DR1, DR15(2), DR4, DR51, DR8 13 37
QQKPGKAPKLLIYAA 7 DQ4, DR1, DR11(5), DR15(2), DR51, DR8 14 40
PGKAPKLLIYAASSL 7 DQ4, DR1, DR11(5), DR4, DR8 15 43 APKLLIYAASSLQSG
15 DR1, DR11(5), DR12(5), DR13(6), DR14(6),DR15(2), DR4 DR51, DR8,
DR9 16 46 LLIYAASSLQSGVPS 15 DR1, DR11(5), DR12(5), DR13(6),
DR14(6),DR15(2), DR4 DR51, DR8, DR9 17 49 YAASSLQSGVPSRFS 1 DR15(2)
18 52 SSLQSGVPSRFSGSG 1 DR15(2) 19 55 GSGVPSRFSGSGSGT 0 20 58
VPSRFSGSGSGTDFT 0 21 61 RFSGSGSGTDFTLTI 0 22 64 GSGSGTDFTLTLSSL 1
DR52 23 67 SGTDFTLTISSLQPE 4 DR4, DR52, DR7, DR9 24 70
DFTLTISSLQPEDFA 4 DQ2, DR4, DR7, DR9 25 73 LTISSLQFEDFATYY 1 DQ2 26
76 SSLQPEDFATYYCQQ 0 27 79 QPEDFATYYCQQSYS 1 DR4 28 82
DFATYYCQQSYSTPP 5 DR4, DR51, DR7 29 85 TYYCQQSYSTPPTFG 4 DR4, DR51,
DR7 30 88 CQQSYSTPPTFGQGT 0 31 91 SYSTPPTFGQGTKVE 0 32 94
TPPTFGQGTKVEIK 0
TABLE-US-00009 TABLE 8 The V.sub.H gene from PG1433 paired with
various light chain genes with differing rates of amino acid
mutation were compared for production levels with the original
clone containingthe IGKV1-39 gene. Number of Light amino acid
concentration IgG name chain gene mutations (.mu.g/ml) PG1433 1-39
0 63, 45.5, 38.6 (avg = 49) PG1631 1-12 4 10.5 PG1632 1-27 7 9.3
PG1634 1D-12 10 10.8 PG1635 1D-33 6 10.2 PG1642 1-5 8 7.1 PG1644
1-9 3 7.8 PG1650 1D-39 3 9.1 PG1652 2D-28 3 7.1 PG1653 3-15 14 7
PG1654 3-20 2 5.2 PG1674 1-40 7 8.2 PG1678 2-11 2 8.1 PG1680 2-14
15 10.8 PG1682 3-1 13 9.9 PG1683 6-57 6 13.9
TABLE-US-00010 TABLE 9 Parameters of stability for stable clones
containing the germline IGKV1-39 gene. IVC at culture days avg pdt
in batch started maximum viable maximum IgG at start previous % at
population cell density % concentration subclone batch run 14 days
.gtoreq.SD avg doublings (.times.10.sup.6 cells/ml) avg (10.sup.9
cells/hr/L) B35.1 21 35 3.5 99 25 3 91 570 40 41 1.3 115 31 3.7 112
568 79 36 0.2 101 62 3.2 97 527 avg 36 3.3 575 B39.4 21 35 1 101 25
2.2 114 424 40 35 0.3 101 25 1.9 98 247 79 34 0.2 59 53 1.7 88 278
avg 35 1.9 316 B38.15 21 35 1.6 106 16 1.5 90 497 40 32 0.3 97 30
3.7 134 557 79 31 0.2 94 63 2.1 76 415 avg 33 3.8 490 B35.30 21 38
5.2 97 25 2.8 81 379 40 51 2.7 131 30 2.7 117 472 79 40 0.7 103 64
1.6 81 325 avg 38 2.0 377 B224.19 23 34 2.6 100 17 3.1 103 507 42
37 0.7 100 33 3.6 120 579 81 38 0.2 100 63 2.3 73 395 avg 38 3.0
402 B224.47 23 32 0.6 102 17 3.5 99 645 42 33 0.3 105 31 3.9 101
378 81 31 0.2 98 64 3.9 101 634 avg 32 3.6 636 B224.53 23 33 0.5
100 17 3.9 110 453 42 32 0.4 97 33 3.7 105 605 81 33 0.3 100 63 3
85 525 avg 33 3.5 962 B224.59 23 39 0.6 104 19 4.3 119 750 42 34
0.2 93 30 4.4 119 779 81 33 0.3 96 61 2.6 67 583 avg 35 3.7 704
B280.3 23 34 0.8 105 17 4.3 105 840 42 32 0.4 98 33 4 98 841 81 31
0.5 95 57 4 95 660 avg 33 4.1 780 B280.12 23 36 1.7 104 15 2 71 426
42 37 0.7 107 30 3.2 116 673 81 33 0.2 96 64 3.1 112 551 avg 35 2.8
550 B280.21 23 32 0.5 102 13 3.2 103 550 42 31 0.4 98 34 3.4 113
592 81 31 0.4 98 65 2.5 83 556 avg 32 3.0 968 B280.36 23 33 1 99 17
3 81 596 42 36 0.5 107 30 4.6 174 1168 81 34 0.3 101 62 3.0 95 835
avg 34 3.7 800 maximum IgG % qab % concentration % correlation
correlation correlation subclone avg (mg/cell/day) avg (mg/L) avg
TF FH TH B35.1 92 9.5 97 122 79 0.95 0.95 0.92 99 10.2 107 188 122
1 0.99 0.99 109 9.4 96 154 100 0.97 0.99 0.98 avg 9.8 155 B39.4 234
16.2 126 141 127 1 0.96 0097 78 12.5 102 56 85 1 3 1 88 9.9 81 57
87 0.59 3 0.93 avg 12.2 111 B38.15 101 7.9 99 97 93 0.99 0.95 0.95
114 7.3 91 118 109 1 0.97 0.97 85 8.8 110 102 98 0.96 0.96 0.99 avg
3 104 B35.30 89 14.5 112 100 71 0.95 2 0.99 125 13.5 107 102 147 1
0.99 0.99 86 10.8 82 114 81 0.98 0.98 0.99 avg 13 140 B224.19 103
19.8 98 207 81 1 0.99 0.99 117 18.1 112 318 124 1 0.94 0.99 80 18.8
90 344 95 1 1 0.99 avg 16.2 257 B224.47 109 22.5 114 387 122 0.98
0.93 0.99 91 20 101 357 112 0.99 0.92 0.95 100 18.8 85 209 66 1
0.99 0.99 avg 15.8 318 B224.53 99 20.6 102 372 154 0.98 0.82 .085
108 24.3 121 379 116 0.98 0.88 0.94 94 15.4 77 231 71 0.99 0.89
0.94 avg 20.2 327 B224.59 107 10.8 105 301 104 0.99 0.78 0.84 111
14.5 95 344 129 0.98 0.92 0.96 81 15.2 99 224 73 0.97 0.99 0.96 avg
19.8 290 B280.3 108 13 109 293 117 0.99 0.98 0.95 108 12.3 103 292
126 0.99 0.98 0.98 85 10.5 88 269 67 0.99 0.98 1 avg 11.5 251
B280.12 77 5.5 95 64 81 0.98 0.98 0.98 122 6.2 101 98 121 1 0.97
0.97 100 5.4 104 78 89 0.98 0.98 0.98 avg 6.1 79 B280.21 97 3.1 128
512 93 0.97 0.92 0.93 104 3.6 51 537 113 3 0.98 0.99 100 8.6 121
114 94 0.97 0.99 1 avg 7.1 121 B280.36 75 10 186 143 159 3 0.99
0.98 146 5.6 104 124 135 1 0.98 0.97 79 0.52 10 8 3 0.97 0.98 1 avg
5.4 92
TABLE-US-00011 TABLE 10 Antibody mixtures used for staining of
lymphocyte populations. Stainings Mixtures Facs Work 1st 2nd 3rd
Final # tubes # Monoclonal dilution volume step step step diltion A
Spleen 1 1-8 CD21.sup.FITC 640 320 0.50 Ckappa rat.sup.PE 160 2.00
CD19.sup.PerCP-Cy5.5 640 0.50 CD23.sup.PE-Cy7 50 1:20 6.40 1000
DAPI Ckappa mouse.sup.BIO-APC 100 1:50 3.20 APC 5000 Clambda
mouse.sup.BIO-APC 100 1:30 3.20 APC 3000 B220.sup.Alex-700 160 2.00
FC block 400 0.80 Spleen 2 9-16 IgD.sup.FITC 640 640 1.00 BM 17-24
Ckappa rat.sup.PE 160 4.00 CD19.sup.PerCP-Cy5.5 500 1.28
IgM.sup.PE-Cy7 640 1.00 DAPI Ckappa mouse.sup.BIO-APC 100 1:50 6.40
APC 5000 Clambda mouse.sup.BIO-APC 100 1:30 6.40 APC 3000
B220.sup.Alex-700 160 4.00 FC block 400 1.60 Spleen 3 25-32 Ckappa
mouse.sup.FITC 400 320 0.80 Ckappa rat.sup.PE 160 2.00
CD19.sup.PerCP-Cy5.5 500 0.64 IgM.sup.PE-Cy7 640 0.50 DAPI Clambda
mouse.sup.BIO-APC 100 1:30 3.20 APC 3000 B220.sup.Alex-700 160 2.00
FC block 400 0.80 Spleen 4 33-40 Ckappa mouse.sup.FITC 400 640 1.60
41-48 lambda.sup.FITC 600 1.07 PP Ckappa rat.sup.PE 160 4.00
CD19.sup.PerCP-Cy5.5 500 1.28 IgM.sup.PE-Cy7 640 1.00 DAPI
IgD.sup.A647 1280 0.50 B220.sup.Alex-700 160 4.00
PNA.sup.BIO-SAV-APC-Cy7 300 2.13 APC-Cy7 FC block 400 1.60 PC 5
49-56 IgM.sup.FITC 160 320 2.00 Ckappa rat.sup.PE 160 2.00
CD19.sup.PerCP-Cy5.5 500 0.64 Ckappa mouse.sup.BIO-PE-Cy7 100 1:50
3.20 PE-Cy7 5000 Clambda mouse.sup.BIO-PE-Cy7 100 1:30 3.20 PE-Cy7
3000 DAPI CD5.sup.APC 320 1.00 B200.sup.Alex-700 160 2.00 FC block
400 0.80 BM 6 57-64 IgM.sup.FITC 160 640 4.00 Ckappa rat.sup.PE 160
4.00 CD19.sup.PerCP-Cy5.5 500 1.28 Ckappa mouse.sup.BIO-PE-Cy7 100
1:50 6.40 PE-Cy7 5000 Clambda mouse.sup.BIO-PE-Cy7 100 1:30 6.40
PE-Cy7 3000 DAPI CD25.sup.APC 80 8.00 B220.sup.Alex-700 160 4.00 FC
block 400 1.60 RAT spleen 7 144 Ckappa rat.sup.PE 160 80 0.5 rat
B220.sup.FITC 160 0.5 Spleen 8 97-104 cyt CD3.sup.FITC 320 320 1
cyt Ckappa rat.sup.PE 80 4.00 cyt CD11c.sup.PE-TexasRED 75 4.27 cyt
NK1.1.sup.BIO-PE-Cy7 200 1.6 PE-Cy7 cyt CD19.sup.PerCP-Cy5.5 320 1
cyt CD4.sup.APC 500 0.64 cyt CD11b.sup.Alex-700 50 6.40 BM = bone
marrow, PC = peritoneal cavity, PP = Peyer's patches.
TABLE-US-00012 TABLE 11 Numbers of lymphocytes harvested from the
bone marrow and spleen of wild-type and transgenic mice *10e6/ml
total vol total cells cells (ml) *10.sup.6 Bone Marrow Wt 18.82
5.05 95.0 Wt 19.24 4.96 95.4 CD19-Cre 23.42 5.08 119.0 CD19-Cre
20.58 4.82 99.2 CD19-Cre 25.77 5.15 132.7 CD19-Cre/HuVk1 17.71 5.06
89.6 CD19-Cre/HuVk1 12.60 5.33 67.2 CD19-Cre/HuVk1 18.13 5.27 95.5
Spleen Wt 41.70 5.36 223.5 Wt 37.85 4.71 178.3 CD19-Cre 60.19 3.77
226.9 CD19-Cre 35.06 3.66 128.3 CD19-Cre 80.69 4.60 371.2
CD19-Cre/HuVk1 51.67 4.48 231.5 CD19-Cre/HuVk1 58.80 6.24 366.9
CD19-Cre/HuVk1 24.37 6.25 152.3
Sequence CWU 1
1
102120DNAArtificial Sequenceprimer 1ccctttccaa tctttatggg
20223DNAArtificial Sequenceprimer 2aggtggattg gtgtcttttt ctc
23322DNAArtificial Sequenceprimer 3gtcatgtcgg cgaccctacg cc
22420DNAArtificial Sequenceprimer 4tgccaggggg aagaccgatg
20538DNAArtificial Sequenceprimer 5gccggccatg gccgaggtrm agcttcagga
gtcaggac 38638DNAArtificial Sequenceprimer 6gccggccatg gccgaggtsc
agctkcagca gtcaggac 38736DNAArtificial Sequenceprimer 7gccggccatg
gcccaggtgc agctgaagsa stcagg 36838DNAArtificial Sequenceprimer
8gccggccatg gccgaggtgc agcttcagga gtcsggac 38938DNAArtificial
Sequenceprimer 9gccggccatg gccgargtcc agctgcaaca gtcyggac
381036DNAArtificial Sequenceprimer 10gccggccatg gcccaggtcc
agctkcagca atctgg 361136DNAArtificial Sequenceprimer 11gccggccatg
gcccagstbc agctgcagca gtctgg 361238DNAArtificial Sequenceprimer
12gccggccatg gcccaggtyc agctgcagca gtctggrc 381336DNAArtificial
Sequenceprimer 13gccggccatg gcccaggtyc agctycagca gtctgg
361439DNAArtificial Sequenceprimer 14gccggccatg gccgaggtcc
arctgcaaca atctggacc 391537DNAArtificial Sequenceprimer
15gccggccatg gcccaggtcc acgtgaagca gtctggg 371635DNAArtificial
Sequenceprimer 16gccggccatg gccgaggtga asstggtgga atctg
351735DNAArtificial Sequenceprimer 17gccggccatg gccgavgtga
agytggtgga gtctg 351838DNAArtificial Sequenceprimer 18gccggccatg
gccgaggtgc agskggtgga gtctgggg 381937DNAArtificial Sequenceprimer
19gccggccatg gccgakgtgc amctggtgga gtctggg 372036DNAArtificial
Sequenceprimer 20gccggccatg gccgaggtga agctgatgga rtctgg
362138DNAArtificial Sequenceprimer 21gccggccatg gccgaggtgc
arcttgttga gtctggtg 382237DNAArtificial Sequenceprimer 22gccggccatg
gccgargtra agcttctcga gtctgga 372336DNAArtificial Sequenceprimer
23gccggccatg gccgaagtga arsttgagga gtctgg 362437DNAArtificial
Sequenceprimer 24gccggccatg gccgaagtga tgctggtgga gtctggg
372538DNAArtificial Sequenceprimer 25gccggccatg gcccaggtta
ctctraaagw gtstggcc 382636DNAArtificial Sequenceprimer 26gccggccatg
gcccaggtcc aactvcagca rcctgg 362735DNAArtificial Sequenceprimer
27gccggccatg gcccaggtyc arctgcagca gtctg 352836DNAArtificial
Sequenceprimer 28gccggccatg gccgatgtga acttggaagt gtctgg
362936DNAArtificial Sequenceprimer 29gccggccatg gccgaggtga
aggtcatcga gtctgg 363045DNAArtificial Sequenceprimer 30cagtcacaga
tcctcgcgaa ttggcccagc cggccatggc csang 453145DNAArtificial
Sequenceprimer 31cagtcacaga tcctcgcgaa ttggcccagc cggccatggc csanc
453237DNAArtificial Sequenceprimer 32gggggtgtcg ttttggctga
ggagacggtg accgtgg 373337DNAArtificial Sequenceprimer 33gggggtgtcg
ttttggctga ggagactgtg agagtgg 373437DNAArtificial Sequenceprimer
34gggggtgtcg ttttggctgc agagacagtg accagag 373537DNAArtificial
Sequenceprimer 35gggggtgtcg ttttggctga ggagacggtg actgagg
373637DNAArtificial Sequenceprimer 36gggggtgtcg ttttggctga
ggagacggtg accgtgg 373737DNAArtificial Sequenceprimer 37gggggtgtcg
ttttggctga ggagacggtg acagtgg 373837DNAArtificial Sequenceprimer
38gggggtgtcg ttttggctga ggagacggtg accagag 373937DNAArtificial
Sequenceprimer 39gggggtgtcg ttttggctga ggagacggtg accgagg
374015PRTArtificial SequenceCDR3 40His Gly Ala Tyr Tyr Thr Tyr Asp
Glu Lys Ala Trp Phe Ala Tyr 1 5 10 15 4115PRTArtificial
SequenceCDR3 41His Gly Ala Phe Tyr Thr Tyr Asp Glu Lys Pro Trp Phe
Ala Tyr 1 5 10 15 4215PRTArtificial SequenceCDR3 42His Ile Ser Tyr
Tyr Arg Tyr Asp Glu Glu Val Ser Phe Ala Tyr 1 5 10 15
437PRTArtificial SequenceCDR3 43Gly Trp Arg Ala Phe Ala Tyr 1 5
4410PRTArtificial SequenceCDR3 44Asp Arg Gly Asn Tyr Tyr Gly Met
Asp Tyr 1 5 10 4512PRTArtificial SequenceCDR3 45Leu Gly Asp Tyr Tyr
Val Asp Trp Phe Phe Ala Val 1 5 10 468PRTArtificial SequenceCDR3
46Asn Phe Pro Ala Trp Phe Ala Phe 1 5 4717PRTArtificial
SequenceCDR3 47Ser Phe Thr Pro Val Pro Phe Tyr Tyr Gly Tyr Asp Trp
Tyr Phe Asp 1 5 10 15 Val 489PRTArtificial SequenceCDR3 48Ser Asp
Tyr Asp Trp Tyr Phe Asp Val 1 5 4910PRTArtificial SequenceCDR3
49Asp Ser Lys Trp Ala Tyr Tyr Phe Asp Tyr 1 5 10 5010PRTArtificial
SequenceCDR3 50Gly Asp Tyr Thr Gly Tyr Gly Met Asp Tyr 1 5 10
5110PRTArtificial SequenceCDR3 51Gly Gly Tyr Asp Gly Tyr Trp Phe
Pro Tyr 1 5 10 5215PRTArtificial Sequenceepitope IGKV1-39 52Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15
5315PRTArtificial Sequenceepitope IGKV1-39 53Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly Asp Arg 1 5 10 15 5415PRTArtificial
Sequenceepitope IGKV1-39 54Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
Asp Arg Val Thr Ile 1 5 10 15 5515PRTArtificial Sequenceepitope
IGKV1-39 55Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys
Arg 1 5 10 15 5615PRTArtificial SequenceEpitope IGKV1-39 56Ala Ser
Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln 1 5 10 15
5715PRTArtificial SequenceEpitope IGKV1-39 57Gly Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser 1 5 10 15 5815PRTArtificial
SequenceEpitope IGKV1-39 58Val Thr Ile Thr Cys Arg Ala Ser Gln Ser
Ile Ser Ser Tyr Leu 1 5 10 15 5915PRTArtificial SequenceEpitope
IGKV1-39 59Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp
Tyr 1 5 10 15 6015PRTArtificial SequenceEpitope IGKV1-39 60Ala Ser
Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys 1 5 10 15
6115PRTArtificial SequenceEpitope IGKV1-39 61Ser Ile Ser Ser Tyr
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys 1 5 10 15 6215PRTArtificial
SequenceEpitope IGKV1-39 62Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys 1 5 10 15 6315PRTArtificial SequenceEpitope
IGKV1-39 63Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 1 5 10 15 6415PRTArtificial SequenceEpitope IGKV1-39 64Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala 1 5 10 15
6515PRTArtificial SequenceEpitope IGKV1-39 65Pro Gly Lys Ala Pro
Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu 1 5 10 15 6615PRTArtificial
SequenceEpitope IGKV1-39 66Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser
Ser Leu Gln Ser Gly 1 5 10 15 6715PRTArtificial SequenceEpitope
IGKV1-39 67Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser 1 5 10 15 6815PRTArtificial SequenceEpitope IGKV1-39 68Tyr Ala
Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 1 5 10 15
6915PRTArtificial SequenceEpitope IGKV1-39 69Ser Ser Leu Gln Ser
Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 1 5 10 15 7015PRTArtificial
SequenceEpitope IGKV1-39 70Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
Ser Gly Ser Gly Thr 1 5 10 15 7115PRTArtificial SequenceEpitope
IGKV1-39 71Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr 1 5 10 15 7215PRTArtificial SequenceEpitope IGKV1-39 72Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 1 5 10 15
7315PRTArtificial SequenceEpitope IGKV1-39 73Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 1 5 10 15 7415PRTArtificial
SequenceEpitope IGKV1-39 74Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro Glu 1 5 10 15 7515PRTArtificial SequenceEpitope
IGKV1-39 75Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe
Ala 1 5 10 15 7615PRTArtificial SequenceEpitope IGKV1-39 76Leu Thr
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr 1 5 10 15
7715PRTArtificial SequenceEpitope IGKV1-39 77Ser Ser Leu Gln Pro
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 1 5 10 15 7815PRTArtificial
SequenceEpitope IGKV1-39 78Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Ser Tyr Ser 1 5 10 15 7915PRTArtificial SequenceEpitope
IGKV1-39 79Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro
Pro 1 5 10 15 8015PRTArtificial SequenceEpitope IGKV1-39 80Thr Tyr
Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro Thr Phe Gly 1 5 10 15
8115PRTArtificial SequenceEpitope IGKV1-39 81Cys Gln Gln Ser Tyr
Ser Thr Pro Pro Thr Phe Gly Gln Gly Thr 1 5 10 15 8215PRTArtificial
SequenceEpitope IGKV1-39 82Ser Tyr Ser Thr Pro Pro Thr Phe Gly Gln
Gly Thr Lys Val Glu 1 5 10 15 8314PRTArtificial SequenceEpitope
IGKV1-39 83Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
1 5 10 84321DNAHomo sapiensCDS(1)..(321) 84gac atc cag atg acc cag
agc ccc agc agc ctg agc gcc agc gtg ggc 48Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 gac aga gtg acc
atc acc tgc aga gcc agc cag agc atc agc agc tac 96Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 ctg aac
tgg tat cag cag aag ccc ggc aag gcc ccc aag ctg ctg atc 144Leu Asn
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45
tac gcc gcc agc tcc ctg cag agc ggc gtg ccc agc aga ttc agc ggc
192Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 agc ggc tcc ggc acc gac ttc acc ctg acc atc agc agc ctg
cag ccc 240Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 gag gac ttc gcc acc tac tac tgc cag cag agc tac
agc acc ccc ccc 288Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr
Ser Thr Pro Pro 85 90 95 acc ttc ggc cag ggc acc aag gtg gag atc
aag 321Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
85107PRTHomo sapiens 85Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 86330DNAHomo
sapiensCDS(1)..(330) 86cag tct gcc ctg acc cag ccc gcc tct gtg tct
ggc agc cct ggc cag 48Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser
Gly Ser Pro Gly Gln 1 5 10 15 agc atc acc atc agc tgc acc ggc acc
agc agc gac gtg ggc ggc tac 96Ser Ile Thr Ile Ser Cys Thr Gly Thr
Ser Ser Asp Val Gly Gly Tyr 20 25 30 aac tac gtg tcc tgg tat cag
cag cac ccc ggc aag gcc ccc aag ctg 144Asn Tyr Val Ser Trp Tyr Gln
Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 atg atc tac gag gtg
tcc aac aga ccc agc ggc gtg agc aac aga ttc 192Met Ile Tyr Glu Val
Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 agc ggc agc
aag agc ggc aac acc gcc agc ctg acc atc agc ggc ctc 240Ser Gly Ser
Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 cag
gct gag gac gag gcc gac tac tac tgc agc agc tac acc agc agc 288Gln
Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90
95 tcc acc ctg gtg ttt ggc ggc gga aca aag ctg acc gtg ctg 330Ser
Thr Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110
87110PRTHomo sapiens 87Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser
Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr
Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln
Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Glu Val
Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser
Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln
Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90
95 Ser Thr Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
110 88321DNARattus norvegicusCDS(1)..(321) 88aga gcc gac gcc gct
ccc acc gtg tcc atc ttc ccc ccc agc atg gaa 48Arg Ala Asp Ala Ala
Pro Thr Val Ser Ile Phe Pro Pro Ser Met Glu 1 5 10 15 cag ctg acc
tct ggc gga gcc acc gtg gtc tgc ttc gtg aac aac ttc 96Gln Leu Thr
Ser Gly Gly Ala Thr Val Val Cys Phe Val Asn Asn Phe 20 25 30 tac
ccc aga gac atc agc gtg aag tgg aag atc gac ggc agc gag cag 144Tyr
Pro Arg Asp Ile Ser Val Lys Trp Lys Ile Asp Gly Ser Glu Gln 35 40
45 agg gac ggc gtg ctg gac agc gtg acc gac cag gac agc aag gac tcc
192Arg Asp Gly Val Leu Asp Ser Val Thr Asp Gln Asp Ser Lys Asp Ser
50 55 60 acc tac agc atg agc agc acc ctg agc ctg acc aag gtg gag
tac gag 240Thr Tyr Ser Met Ser Ser Thr Leu Ser Leu Thr Lys Val Glu
Tyr Glu 65 70 75 80 agg cac aac ctg tac acc tgc gag gtg gtg cac aag
acc agc tcc agc 288Arg His Asn Leu Tyr Thr Cys Glu Val Val His Lys
Thr Ser Ser Ser 85 90 95 ccc gtg gtc aag tcc ttc aac cgg aac gag
tgt 321Pro Val Val Lys Ser Phe Asn Arg Asn Glu Cys 100 105
89107PRTRattus norvegicus 89Arg Ala Asp Ala Ala Pro Thr Val Ser Ile
Phe Pro Pro Ser Met Glu 1 5 10 15 Gln Leu Thr Ser Gly Gly Ala Thr
Val Val Cys Phe Val Asn Asn Phe 20 25 30 Tyr Pro Arg Asp Ile Ser
Val Lys Trp Lys Ile Asp Gly Ser Glu Gln 35 40 45 Arg Asp Gly Val
Leu Asp Ser Val Thr Asp Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr
Ser Met Ser Ser Thr Leu Ser Leu Thr Lys Val Glu Tyr Glu 65 70
75 80 Arg His Asn Leu Tyr Thr Cys Glu Val Val His Lys Thr Ser Ser
Ser 85 90 95 Pro Val Val Lys Ser Phe Asn Arg Asn Glu Cys 100 105
90865DNAArtificial SequenceIGKV1-39/J-Ck 90ggtaccgcgg ccgccaccat
ggacatgaga gtgcccgccc agctcctggg gctcctgcta 60ctctggctcc gaggtaagga
tggagaacac taggaattta ctcagccagt gtgctcagta 120ctgactggaa
cttcagggaa gttctctgat aacatgatta atagtaagaa tatttgtttt
180tatgtttcca atctcaggtg ccagatgtga catccagatg acccagagcc
ccagcagcct 240gagcgccagc gtgggcgaca gagtgaccat cacctgcaga
gccagccaga gcatcagcag 300ctacctgaac tggtatcagc agaagcccgg
caaggccccc aagctgctga tctacgccgc 360cagctccctg cagagcggcg
tgcccagcag attcagcggc agcggctccg gcaccgactt 420caccctgacc
atcagcagcc tgcagcccga ggacttcgcc acctactact gccagcagag
480ctacagcacc ccccccacct tcggccaggg caccaaggtg gagatcaaga
gagccgacgc 540cgctcccacc gtgtccatct tcccccccag catggaacag
ctgacctctg gcggagccac 600cgtggtctgc ttcgtgaaca acttctaccc
cagagacatc agcgtgaagt ggaagatcga 660cggcagcgag cagagggacg
gcgtgctgga cagcgtgacc gaccaggaca gcaaggactc 720cacctacagc
atgagcagca ccctgagcct gaccaaggtg gagtacgaga ggcacaacct
780gtacacctgc gaggtggtgc acaagaccag ctccagcccc gtggtcaagt
ccttcaaccg 840gaacgagtgt tgagctagcg agctc 86591874DNAArtificial
SequenceIGLV2-14/J-Ck 91ggtaccgcgg ccgccaccat ggacatgaga gtgcccgccc
agctcctggg gctcctgcta 60ctctggctcc gaggtaagga tggagaacac taggaattta
ctcagccagt gtgctcagta 120ctgactggaa cttcagggaa gttctctgat
aacatgatta atagtaagaa tatttgtttt 180tatgtttcca atctcaggtg
ccagatgtca gtctgccctg acccagcccg cctctgtgtc 240tggcagccct
ggccagagca tcaccatcag ctgcaccggc accagcagcg acgtgggcgg
300ctacaactac gtgtcctggt atcagcagca ccccggcaag gcccccaagc
tgatgatcta 360cgaggtgtcc aacagaccca gcggcgtgag caacagattc
agcggcagca agagcggcaa 420caccgccagc ctgaccatca gcggcctcca
ggctgaggac gaggccgact actactgcag 480cagctacacc agcagctcca
ccctggtgtt tggcggcgga acaaagctga ccgtgctgag 540agccgacgcc
gctcccaccg tgtccatctt cccccccagc atggaacagc tgacctctgg
600cggagccacc gtggtctgct tcgtgaacaa cttctacccc agagacatca
gcgtgaagtg 660gaagatcgac ggcagcgagc agagggacgg cgtgctggac
agcgtgaccg accaggacag 720caaggactcc acctacagca tgagcagcac
cctgagcctg accaaggtgg agtacgagag 780gcacaacctg tacacctgcg
aggtggtgca caagaccagc tccagccccg tggtcaagtc 840cttcaaccgg
aacgagtgtt gagctagcga gctc 8749213373DNAArtificial
SequenceVkP-IGKV1-39/J-Ck 92ggccggccca catgaaacaa tgggaaccat
gtgacaatca cagaggtgtt gttactatag 60caaaagggat tgttactctc cacatccctt
taagtaactt gaaggcctga tagacccacc 120ctctaagact tcattagaca
ttccctacga atggttatac tctcctgtat actcccaata 180caactctaaa
atatattatt ccatatagtc cttaggtttg tattaaagtt tgactttttt
240ccttcaaaat atctcttgtc acaacagcgg ctctagagag aaatacattc
cctccaggca 300aatctatgct gcgctggtct gacctgggac cctggggaca
ttgcccctgt gctgagttac 360taagatgagc cagccctgca gctgtgctca
gcctgcccca tgccctgctg attgatttgc 420atgttccaga gcacagcccc
ctgccctgaa gactttttta tgggctggtc gcaccctgtg 480caggagtcag
tctcagtcag gagccaccat ggacatgaga gtgcccgccc agctcctggg
540gctcctgcta ctctggctcc gaggtaagga tggagaacac taggaattta
ctcagccagt 600gtgctcagta ctgactggaa cttcagggaa gttctctgat
aacatgatta atagtaagaa 660tatttgtttt tatgtttcca atctcaggtg
ccagatgtga catccagatg acccagagcc 720ccagcagcct gagcgccagc
gtgggcgaca gagtgaccat cacctgcaga gccagccaga 780gcatcagcag
ctacctgaac tggtatcagc agaagcccgg caaggccccc aagctgctga
840tctacgccgc cagctccctg cagagcggcg tgcccagcag attcagcggc
agcggctccg 900gcaccgactt caccctgacc atcagcagcc tgcagcccga
ggacttcgcc acctactact 960gccagcagag ctacagcacc ccccccacct
tcggccaggg caccaaggtg gagatcaaac 1020gtaagtacac ttttctcatc
tttttttatg tgtaagacac aggttttcat gttaggagtt 1080aaagtcagtt
cagaaaatct tgagaaaatg gagagggctc attatcagtt gacgtggcat
1140acagtgtcag attttctgtt tatcaagcta gtgagattag gggcaaaaag
aggctttagt 1200tgagaggaaa gtaattaata ctatggtcac catccaagag
attggatcgg agaataagca 1260tgagtagtta ttgagatctg ggtctgactg
caggtagcgt ggtcttctag acgtttaagt 1320gggagatttg gaggggatga
ggaatgaagg aacttcagga tagaaaaggg ctgaagtcaa 1380gttcagctcc
taaaatggat gtgggagcaa actttgaaga taaactgaat gacccagagg
1440atgaaacagc gcagatcaaa gaggggcctg gagctctgag aagagaagga
gactcatccg 1500tgttgagttt ccacaagtac tgtcttgagt tttgcaataa
aagtgggata gcagagttga 1560gtgagccgta ggctgagttc tctcttttgt
ctcctaagtt tttatgacta caaaaatcag 1620tagtatgtcc tgaaataatc
attaagctgt ttgaaagtat gactgcttgc catgtagata 1680ccatggcttg
ctgaataatc agaagaggtg tgactcttat tctaaaattt gtcacaaaat
1740gtcaaaatga gagactctgt aggaacgagt ccttgacaga cagctcaagg
ggtttttttc 1800ctttgtctca tttctacatg aaagtaaatt tgaaatgatc
ttttttatta taagagtaga 1860aatacagttg ggtttgaact atatgtttta
atggccacgg ttttgtaaga catttggtcc 1920tttgttttcc cagttattac
tcgattgtaa ttttatatcg ccagcaatgg actgaaacgg 1980tccgcaacct
cttctttaca actgggtgac ctcgcggctg tgccagccat ttggcgttca
2040ccctgccgct aagggccatg tgaacccccg cggtagcatc ccttgctccg
cgtggaccac 2100tttcctgagg cacagtgata ggaacagagc cactaatctg
aagagaacag agatgtgaca 2160gactacacta atgtgagaaa aacaaggaaa
gggtgactta ttggagattt cagaaataaa 2220atgcatttat tattatattc
ccttatttta attttctatt agggaattag aaagggcata 2280aactgcttta
tccagtgtta tattaaaagc ttaatgtata taatctttta gaggtaaaat
2340ctacagccag caaaagtcat ggtaaatatt ctttgactga actctcacta
aactcctcta 2400aattatatgt catattaact ggttaaatta atataaattt
gtgacatgac cttaactggt 2460taggtaggat atttttcttc atgcaaaaat
atgactaata ataatttagc acaaaaatat 2520ttcccaatac tttaattctg
tgatagaaaa atgtttaact cagctactat aatcccataa 2580ttttgaaaac
tatttattag cttttgtgtt tgacccttcc ctagccaaag gcaactattt
2640aaggaccctt taaaactctt gaaactactt tagagtcatt aagttattta
accactttta 2700attactttaa aatgatgtca attccctttt aactattaat
ttattttaag gggggaaagg 2760ctgctcataa ttctattgtt tttcttggta
aagaactctc agttttcgtt tttactacct 2820ctgtcaccca agagttggca
tctcaacaga ggggactttc cgagaggcca tctggcagtt 2880gcttaagatc
agaagtgaag tctgccagtt cctcccaggc aggtggccca gattacagtt
2940gacctgttct ggtgtggcta aaaattgtcc catgtggtta caaaccatta
gaccagggtc 3000tgatgaattg ctcagaatat ttctggacac ccaaatacag
accctggctt aaggccctgt 3060ccatacagta ggtttagctt ggctacacca
aaggaagcca tacagaggct aatatcagag 3120tattcttgga agagacagga
gaaaatgaaa gccagtttct gctcttacct tatgtgcttg 3180tgttcagact
cccaaacatc aggagtgtca gataaactgg tctgaatctc tgtctgaagc
3240atggaactga aaagaatgta gtttcaggga agaaaggcaa tagaaggaag
cctgagaata 3300tcttcaaagg gtcagactca atttactttc taaagaagta
gctaggaact agggaataac 3360ttagaaacaa caagattgta tatatgtgca
tcctggcccc attgttcctt atctgtaggg 3420ataagcgtgc ttttttgtgt
gtctgtatat aacataactg tttacacata atacactgaa 3480atggagccct
tccttgttac ttcataccat cctctgtgct tccttcctca ggggccgacg
3540ccgctcccac cgtgtccatc ttccccccca gcatggaaca gctgacctct
ggcggagcca 3600ccgtggtctg cttcgtgaac aacttctacc ccagagacat
cagcgtgaag tggaagatcg 3660acggcagcga gcagagggac ggcgtgctgg
acagcgtgac cgaccaggac agcaaggact 3720ccacctacag catgagcagc
accctgagcc tgaccaaggt ggagtacgag aggcacaacc 3780tgtacacctg
cgaggtggtg cacaagacca gctccagccc cgtggtcaag tccttcaacc
3840ggaacgagtg ttgaagacaa aggtcctgag acgccaccac cagctcccca
gctccatcct 3900atcttccctt ctaaggtctt ggaggcttcc ccacaagcga
cctaccactg ttgcggtgct 3960ccaaacctcc tccccacctc cttctcctcc
tcctcccttt ccttggcttt tatcatgcta 4020atatttgcag aaaatattca
ataaagtgag tctttgcact tgagatctct gtctttctta 4080ctaaatggta
gtaatcagtt gtttttccag ttacctgggt ttctcttcta aagaagttaa
4140atgtttagtt gccctgaaat ccaccacact taaaggataa ataaaaccct
ccacttgccc 4200tggttggctg tccactacat ggcagtcctt tctaaggttc
acgagtacta ttcatggctt 4260atttctctgg gccatggtag gtttgaggag
gcatacttcc tagttttctt cccctaagtc 4320gtcaaagtcc tgaaggggga
cagtctttac aagcacatgt tctgtaatct gattcaacct 4380acccagtaaa
cttggcgaag caaagtagaa tcattatcac aggaagcaaa ggcaacctaa
4440atgtgcaagc aataggaaaa tgtggaagcc catcatagta cttggacttc
atctgctttt 4500gtgccttcac taagttttta aacatgagct ggctcctatc
tgccattggc aaggctgggc 4560actacccaca acctacttca aggacctcta
taccgtgaga ttacacacat acatcaaaat 4620ttgggaaaag ttctaccaag
ctgagagctg atcaccccac tcttaggtgc ttatctctgt 4680acaccagaaa
ccttaagaag caaccagtat tgagagactc atttatgaaa gtctaaaact
4740ggatacaacc aaaatgtcca ccaacagtta aattatgaca tgttcacaat
tgagctatta 4800cttaataagg agaattaata aaataaaact taagagcata
gtttaatctc ataaacaaga 4860taataagcaa aacaaaacat tttttcatcc
atgtaagttt aaaagcaggt aaaatttaaa 4920attaagagag acataagttt
tgaggtagca agatggaaac tctggggctt ggggaatgtt 4980ctgtctctct
gtatgggatg tgaaagttac tattgtggaa ttgggatcta tgttcttcct
5040gtatatattg tatacttcat aataacttca cctaaagaaa tatctaatac
ccagtgcata 5100cataaaagag gatacaagga atgaatcata cgtcaaggcc
agaaagacaa taaagtaggg 5160gatccaggat caaatctccc acaaccttga
gccttctact attctgcctt ccagagctca 5220aagtacaaaa cacataattc
aaacacatga tccctccttg gggtctcttc cttcatgcat 5280cgaattagaa
atagccatgt ataaaatgag atagaagaga ccttcatcaa caggtcaaag
5340aatataggta attttgtctg ggtatgaaga gcccacgtat caaaggttac
attagggaag 5400gaagaggaca ctaacagtga ctttcattct ccccctcttc
ctggaggccc ctgcatttag 5460tccctcgtgg gctcatccac tcagcacaca
tttactaagc atcttctcag cctacactct 5520gaaggcagtg cagaataatg
ttagtgtccc ttcccccagt taatatgcag tccagtttcc 5580ctgctccttc
cctttctcag tccacataag gatgatggga aaggacagtc accaaatagg
5640agagggcaac cctttgcctt cctacctctt gagaatgtac attattatcc
actttttgaa 5700acttctttta attgcttttt tttaatttgt cttttcaaat
agcataacct tgttcatcca 5760tttctgggaa ccaaatttat caatcaacag
tgcctctaat ctggctatta atacaaaaat 5820gcctcctcaa aatatatatg
ttcgagtctt atctaaaaca gaacccacaa taaaaaagaa 5880gaaagaatac
atataagcat ttatataatt ctgagcaacc ttgtgctttg tgaaaaaaat
5940ataatctaat gtcacatgct gtattctttt tatttaacac tggtgaaatt
ataccattag 6000agagaaagag gacagatcac tgatcctagg atctagggat
gttacagata agaaaacaaa 6060tgtgacaaag agctgtcaca aggaggatct
tcaaggtcac agaatcactg tcttgatttc 6120agtggtggtt acatacattt
aaatatgtga taaaatgttg ttgaactata ttcatatatt 6180gtaccaatgt
caaatgctta attttggctc tatagtataa ttatgcacta aataactatt
6240tggacaaaga aaatgatgtt tacatcaaag gtgaggccat atttgttagg
aacataactt 6300aaaaaccatt ttggataact aatgaaaagc cattttgtgt
gccttggcat atcatgccta 6360agctgtcacc agatagatct aataagacct
aagcctcaga agcaagcccc tgcccagcaa 6420gcaggcagca cagataagag
ctaaacccag gacaggccat gatatgctaa tgaactacct 6480tcaaggtggt
gttgctgacc tagtgaacca gccccaagct gtgagcccca atagcacaaa
6540gctactgccc aaagaaatta tacaaaaatt ggaactttgg gaatggtgtg
caggatcgct 6600ctgctgtatg cctggaacac agcttctcta tgttttgtat
tgataccagt ctagaagctt 6660ccaaaacttt ctcactgaag aagattcccc
atgtgggacc cctacagact cttttgccca 6720aacaactgct tccctcctgg
tgtgatatct gttttgcttt tatgttagca taatattata 6780aggaatgttt
gtgtgaataa accaaacata ttttaaaagc aaatattgta tgcacatcct
6840aattgctaaa aagtttacag ctaatagtcc catgctctcc acaatactgg
atccaaataa 6900gtcctaattt caatgttggg catctttaca gagagaaaga
cattaaaaat gaagagacat 6960gcagagagtg caccatgcca tcgtggagac
agactgaagt gacacaactg ttagtcaaag 7020aggattaagg acttccagaa
gccaccaaag gaaggaggta tgaagtggtt tctccctcag 7080agtatccaga
ggagactaaa ccaaccaaca cctttttgct taagacttct tgccttcagg
7140actgtgagaa ggtagcttcc tattgttcta agccccagta tgtggcattt
tgttaaggta 7200gagtcaagaa accaataaaa tgcagacaga caaaaggata
gctgagtttt ccaggccctt 7260ccttcttatt tttggttttg ttggtggtgg
tggtggtggt ggtgatggtg gtggttttgt 7320ttatgttttg tttggggagt
tttttggggt ttttttgggt tttgtttttg ttgttgtttt 7380gggggttttt
gttgttgttg ttgtttgctt ttttgttttt tgttttttgt ttttttgaga
7440cagtgtttct ctgtatagcc ctggctgtcc tggagttcct tctatctcta
atgtctacat 7500ctcagagggg atcctctaat ttcaaatgag cagtagctct
ccatttttag ctcttattta 7560ttcatttatt tacttactta cttattgtct
gtagatgaaa gaattttgga gtgggaaagg 7620gttcatgagc ccccagcaac
taatgaggag ctacagacaa ttgatgtttc tggggaaagg 7680agactcagtt
tctttgagag tatagcttct gatgggtcaa ccatgttcct gtggctgatg
7740tcacacccag gagtatgcag acaacagaaa ctggagttaa tgagttgttt
taaaaataaa 7800aaagggcatg aagcttggga tagaaattaa ggataaatac
aattaaatac aggaaattct 7860gaaagaatta ataaaaacat ttcttttttt
aaaaaaaaat ccagaattag ctatgcttct 7920tcaaaattgc ttctggagaa
ctttacaagt taaataagtt atattgtaga aaaggtagag 7980aggagaatag
tggaagagag agataaggag acttcaaaag gagtggaggg agatagagga
8040ggagaaagca gaagcaatgg ctgatagaca caggataaga gggaacagaa
aggagaaaga 8100ggaagccagg atgggtattt ctttgcctat ctgtgacttg
cacatggtct tggcaattat 8160tgatgagttc aaggcttaat tcttcacttg
tgccaactca acagagtctt tctttcttat 8220aaccaggccc ccagtatgct
catgtatgta tcaggtcctc ttatctcctt atagcaatcc 8280tgtttataac
tgggtaactt tgtgaaggga aggaagtgca cactgagatg tgctacaact
8340ttttaataca aaattttgaa gagtttgtac aatgtatgta taattaataa
ttaatattat 8400gcactttaga ttttgatttc aactcaagat actaattcta
tatatatggg ttaaatcaat 8460atattaataa gtttaatttc acatgcttat
ttttattgtg gttttcgaga cagggtttct 8520ctgtatagcc ctggctgtcc
tggaacccac tttgtagacc aggctggcct caaactcaga 8580aacctacctg
cctctgcctc tgcctctgcc tctgcctctg cctctgcctc tgcctctgcc
8640tctgcctctg cctctgcctc tgcctctgcc tctgcctctg cctctgcctc
tgcctctgcc 8700tctgcctctg cctctgcctc tgcctctgcc tctgcctctg
cctctgcctc tgcctctgcc 8760tctgcctagt gctggaatta aaggtttgcg
ccaccacgcc cggtgaaatt tttaaacttt 8820atatatgtct cattctattt
ctatcagata ggactgtgta gactgtgcta aactaataaa 8880tgtgccctca
aaagtaatcg caagttgtat tgttgttgtt ttgctttgct ttgctttgct
8940ttgctttgct ttgctttgct ttgctttgct ttgctttgct ttgctttgct
ttgctttgct 9000ttgctttgct ttgctttgct ttgctttttt gttttgggtt
tttttccggg ggagggaggg 9060tggagaaaga atcttactat gaagctctga
ctgtcctggg aactcactat atagatcagg 9120cttgattcaa ctcatagaga
tctgccttct tctgcctccc aagtgctggg aataaaggca 9180tacacctcca
tgcccagata gtgatcccaa gttttagcaa aagtttctag acttgacatt
9240aatcgatgga gatagacatg aattacacaa agaactaatg tggagtttac
ctgaatcata 9300ctctatactt tatcagagat taaattaaca tttaataatc
cagtgccagg ctagaggcac 9360cattcaatgg cagtgtttgc catcatgcat
aggcttagtc ttcagtgctg aaaggcattg 9420ggggcaatat tactcattat
acagatgaga aactgggaaa gacttgcctc agattctcta 9480ctgaaaggct
gagtttgtgg cttctagaaa atcttttact ttcaatattt ttaatgtata
9540atttttttat ttccactgat tttatttttt atttttaaca tttataagaa
ataaatgcaa 9600taaaccaaat acatggacaa aaaaatacaa gaatcatatg
atcacctcaa tggaaggaaa 9660aaaaaagaaa gaaaaagtct ttgataagat
tcaacattca ttcttttttt attagatatt 9720ttcttcattt acatttcaaa
tgctatcccc aaagccccct ataccttccc ctgccctgct 9780ccccaaccca
cccactcctg ctttctggcc ctggcattcc tctgtactga ggcatatgat
9840cttcaaaaaa ccaagggcct ctcctctcat tggtggccga ctattaggcc
atcttttgct 9900acatatgcaa ctagagacac agctctgggg gttactggtt
agttcatatt gttagtcctc 9960ctatagagtt gcagacccct ttagctcctt
ggatactttc tctagttcct tcattagggg 10020ccctgtgtcc catccaatag
atgactgtga gcatccactt ctgtatttgc caggcactgg 10080catagcctca
cgagaaagag agagctatgt caggatcctg tcagtaaaat ctttctggca
10140tatgcaatag tatctgggtt tggtggttgt atatgggatg gatccccaag
tggagcagtc 10200tctgaatggt ccttccttcc atctcagctc caaactttgt
ctctataact ccttccatgg 10260gtattttgtt ccccattcta agaaggagtg
aagaatccac actttggtct tccttcttct 10320tgagtttcat atgttgcatc
ttggatattc taagtttctg ggttaatatc cacgtatcag 10380tgagtgcata
tcatgcgtgt tattttgtga ttagtttacc tcactcagga tgatatcctc
10440cagatgcatc catttgccta agaatttcat taattcactg tttttaattg
ctgaatagta 10500ctccattgtg taaatgtacc acattttctg tatccattcc
tctgttgagg ggcatctggg 10560ttctttccag cttctggcta ttataaataa
ggctgctatg agcatagcgg agcatgtgtc 10620cttatcaagt tggaacatct
tctaggtata tgcccaggag aggaattgct ggatcttccg 10680gtagtaccat
caacatgcat tcttaataaa agccctagaa caaggaggac tgtaggaaac
10740atattccaac ataataaagg ttatgtatga caaactcatg accaatatca
tcctaaatga 10800atgaaaccat taataagctc cattaaaatc agaggactgc
ccactatccc tacttctcat 10860ccataatgag attgaagcat tagctggagc
aataaggcaa gagaagggat acaaatggga 10920aaatattaag tcaaattgtt
ttcaattgaa gattatatta tcttataccc aatgacctca 10980aattttgact
agaaaaattg tagaaattat caataatttc agcaaagtgt tatgatgcac
11040cacatcctta ttcttctccc cagcttctgc ttgcttctct cttcttgctc
ttcatccttt 11100ctgtccttcc atctgcctgc actcttgtct caagactgag
tgcagcgtgt aactctcctg 11160tgactgagta tctcacaaaa cgttctacct
gccaaacctg gatgagccct ttgtctttct 11220gaagctatga ggctctctac
atagactcaa gaaggaaatg acagggagga ggtaataatg 11280aagtggggaa
ggctgacatt agcattgctc ctgtgtggct ccttaatttc tcatacttca
11340cactgagatg ttattaactg tgactcatag gtgaagaagc cagagctaag
gttctcatat 11400ttgagtgtta tagaatgagt agagcagtag ttctcaaact
atgggtcatg actcctttat 11460gggtcaaact accctttcac acaggttgca
tatcagatat cctaatttta tatacatata 11520tatatgcata tgtatatata
tatatttcac aacagtagga aaattattta gtaatcattt 11580tatagttgtg
ggtcatggca acatgaggaa ctgtattaaa gggttgcagc attaggaatg
11640ttgagaccca ctgtaataga gaatgaggct taaggcaggg ctataaagcc
caatggacca 11700tgtgcctttt ccaacatttg ccacatggta agctctgtat
agacttttta aagaacattg 11760gtttgtaatt ttaaatggat aagggtcttc
actgtctatc acccatctat ataataaata 11820cataagtttt gattccacca
tggattcaaa tgcaaaaatc ctcaacctaa gacatagcag 11880tgaaacattg
atgaccaaat aggaaatcca tgtagagacc ttctatcttc tgatggctcc
11940acaggcacca tcttgcaaca gagttctact ttgctaccag taatgaatac
agtgtctcaa 12000ctcctgccat tgaatcttca ggaagcccct gaaatgactt
gtactacacc atttcttaaa 12060gacagaaaag ctaagactta gagggaataa
atgtcatgcc tgagatcatg caaccaatta 12120agtccaactt ggcctgatca
agaggcacaa ttcaaaagca atgttgttcc ttcactagct 12180cttgtgtatg
gttgctgatt ccggaagcaa agtatcagtg aatatcccta gtgggaaaag
12240acttggaaat caaatgtctc atttaacaga ttaggagatg aaacggtaga
ctctgtgtag 12300ttgtacaccc ctgtgatccc atcgctagga agactgaggc
aggaagtcct cgagctcaaa 12360ccagcttagg ctacacagag aaactatcta
aaaaataatt actaactact taataggaga 12420ttggatgtta agatctggtc
actaagaggc agaattgaga ttcgaagcca gtattttcta 12480cctggtatgt
tttaaattgc agtaaggatc taagtgtaga tatataataa taagattcta
12540ttgatctctg caacaacaga gagtgttaga tttgtttgga aaaaaatatt
atcagccaac 12600atcttctacc atttcagtat agcacagagt acccacccat
atctccccac ccatccccca 12660taccagactg gttattgatt ttcatggtga
ctggcctgag aagattaaaa aaagtaatgc 12720taccttattg ggagtgtccc
atggaccaag atagcaactg tcatagctac cgtcacactg 12780ctttgatcaa
gaagaccctt tgaggaactg aaaacagaac cttaggcaca tctgttgctt
12840tcgctcccat cctcctccaa cagcctgggt ggtgcactcc acaccctttc
aagtttccaa 12900agcctcatac acctgctccc taccccagca cctggccaag
gctgtatcca gcactgggat 12960gaaaatgata ccccacctcc atcttgtttg
atattactct atctcaagcc ccaggttagt 13020ccccagtccc aatgcttttg
cacagtcaaa actcaacttg gaataatcag tatccttgaa 13080gagttctgat
atggtcactg ggcccatata ccatgtaaga catgtggaaa agatgtttca
13140tggggcccag acacgttcta gaagtacctg agagtggcaa aaaatagttg
tgctaaatag 13200tttggccatc tttaggctga gagactagga aatacagcga
tggactatat cagcattgca 13260ggatagttgt cagtaaacac cccacaaccc
ataacagaag tattctcttc tttctatatc 13320ccttttccat ccatgtagat
ggctgtcttc atatttgttc tagacggccg gcc 133739312892DNAArtificial
SequenceVkP-IGKV1-39/J-Ck-delta1 93ggccggccca catgaaacaa tgggaaccat
gtgacaatca cagaggtgtt gttactatag 60caaaagggat tgttactctc cacatccctt
taagtaactt gaaggcctga tagacccacc 120ctctaagact tcattagaca
ttccctacga atggttatac tctcctgtat actcccaata 180caactctaaa
atatattatt ccatatagtc cttaggtttg tattaaagtt tgactttttt
240ccttcaaaat atctcttgtc acaacagcgg ctctagagag aaatacattc
cctccaggca 300aatctatgct gcgctggtct gacctgggac cctggggaca
ttgcccctgt gctgagttac 360taagatgagc cagccctgca gctgtgctca
gcctgcccca tgccctgctg attgatttgc 420atgttccaga gcacagcccc
ctgccctgaa gactttttta tgggctggtc gcaccctgtg 480caggagtcag
tctcagtcag gagccaccat ggacatgaga gtgcccgccc agctcctggg
540gctcctgcta ctctggctcc gaggtaagga tggagaacac taggaattta
ctcagccagt 600gtgctcagta ctgactggaa cttcagggaa gttctctgat
aacatgatta atagtaagaa 660tatttgtttt tatgtttcca atctcaggtg
ccagatgtga catccagatg acccagagcc 720ccagcagcct gagcgccagc
gtgggcgaca gagtgaccat cacctgcaga gccagccaga 780gcatcagcag
ctacctgaac tggtatcagc agaagcccgg caaggccccc aagctgctga
840tctacgccgc cagctccctg cagagcggcg tgcccagcag attcagcggc
agcggctccg 900gcaccgactt caccctgacc atcagcagcc tgcagcccga
ggacttcgcc acctactact 960gccagcagag ctacagcacc ccccccacct
tcggccaggg caccaaggtg gagatcaaac 1020gtaagtacac ttttctcatc
tttttttatg tgtaagacac aggttttcat gttaggagtt 1080aaagtcagtt
cagaaaatct tgagaaaatg gagagggctc attatcagtt gacgtggcat
1140acagtgtcag attttctgtt tatcaagcta gtgagattag gggcaaaaag
aggctttagt 1200tgagaggaaa gtaattaata ctatggtcac catccaagag
attggatcgg agaataagca 1260tgagtagtta ttgagatctg ggtctgactg
caggtagcgt ggtcttctag acgtttaagt 1320gggagatttg gaggggatga
ggaatgaagg aacttcagga tagaaaaggg ctgaagtcaa 1380gttcagctcc
taaaatggat gtgggagcaa actttgaaga taaactgaat gacccagagg
1440atgaaacagc gcagatcaaa gaggggcctg gagctctgag aagagaagga
gactcatccg 1500tgttgagttt ccacaagtac tgtcttgagt tttgcaataa
aagtgggata gcagagttga 1560gtgagccgta ggctgagttc tctcttttgt
ctcctaagtt tttatgacta caaaaatcag 1620tagtatgtcc tgaaataatc
attaagctgt ttgaaagtat gactgcttgc catgtagata 1680ccatggcttg
ctgaataatc agaagaggtg tgactcttat tctaaaattt gtcacaaaat
1740gtcaaaatga gagactctgt aggaacgagt ccttgacaga cagctcaagg
ggtttttttc 1800ctttgtctca tttctacatg aaagtaaatt tgaaatgatc
ttttttatta taagagtaga 1860aatacagttg ggtttgaact atatgtttta
atggccacgg ttttgtaaga catttggtcc 1920tttgttttcc cagttattac
tcgattgtaa ttttatatcg ccagcaatgg actgaaacgg 1980tccgcaacct
cttctttaca actgggtgac ctcgcggctg tgccagccat ttggcgttca
2040ccctgccgct aagggccatg tgaacccccg cggtagcatc ccttgctccg
cgtggaccac 2100tttcctgagg cacagtgata ggaacagagc cactaatctg
aagagaacag agatgtgaca 2160gactacacta atgtgagaaa aacaaggaaa
gggtgactta ttggagattt cagaaataaa 2220atgcatttat tattatattc
ccttatttta attttctatt agggaattag aaagggcata 2280aactgcttta
tccagtgtta tattaaaagc ttaatgtata taatctttta gaggtaaaat
2340ctacagccag caaaagtcat ggtaaatatt ctttgactga actctcacta
aactcctcta 2400aattatatgt catattaact ggttaaatta atataaattt
gtgacatgac cttaactggt 2460taggtaggat atttttcttc atgcaaaaat
atgactaata ataatttagc acaaaaatat 2520ttcccaatac tttaattctg
tgatagaaaa atgtttaact cagctactat aatcccataa 2580ttttgaaaac
tatttatttg gctacaccaa aggaagccat acagaggcta atatcagagt
2640attcttggaa gagacaggag aaaatgaaag ccagtttctg ctcttacctt
atgtgcttgt 2700gttcagactc ccaaacatca ggagtgtcag ataaactggt
ctgaatctct gtctgaagca 2760tggaactgaa aagaatgtag tttcagggaa
gaaaggcaat agaaggaagc ctgagaatat 2820cttcaaaggg tcagactcaa
tttactttct aaagaagtag ctaggaacta gggaataact 2880tagaaacaac
aagattgtat atatgtgcat cctggcccca ttgttcctta tctgtaggga
2940taagcgtgct tttttgtgtg tctgtatata acataactgt ttacacataa
tacactgaaa 3000tggagccctt ccttgttact tcataccatc ctctgtgctt
ccttcctcag gggccgacgc 3060cgctcccacc gtgtccatct tcccccccag
catggaacag ctgacctctg gcggagccac 3120cgtggtctgc ttcgtgaaca
acttctaccc cagagacatc agcgtgaagt ggaagatcga 3180cggcagcgag
cagagggacg gcgtgctgga cagcgtgacc gaccaggaca gcaaggactc
3240cacctacagc atgagcagca ccctgagcct gaccaaggtg gagtacgaga
ggcacaacct 3300gtacacctgc gaggtggtgc acaagaccag ctccagcccc
gtggtcaagt ccttcaaccg 3360gaacgagtgt tgaagacaaa ggtcctgaga
cgccaccacc agctccccag ctccatccta 3420tcttcccttc taaggtcttg
gaggcttccc cacaagcgac ctaccactgt tgcggtgctc 3480caaacctcct
ccccacctcc ttctcctcct cctccctttc cttggctttt atcatgctaa
3540tatttgcaga aaatattcaa taaagtgagt ctttgcactt gagatctctg
tctttcttac 3600taaatggtag taatcagttg tttttccagt tacctgggtt
tctcttctaa agaagttaaa 3660tgtttagttg ccctgaaatc caccacactt
aaaggataaa taaaaccctc cacttgccct 3720ggttggctgt ccactacatg
gcagtccttt ctaaggttca cgagtactat tcatggctta 3780tttctctggg
ccatggtagg tttgaggagg catacttcct agttttcttc ccctaagtcg
3840tcaaagtcct gaagggggac agtctttaca agcacatgtt ctgtaatctg
attcaaccta 3900cccagtaaac ttggcgaagc aaagtagaat cattatcaca
ggaagcaaag gcaacctaaa 3960tgtgcaagca ataggaaaat gtggaagccc
atcatagtac ttggacttca tctgcttttg 4020tgccttcact aagtttttaa
acatgagctg gctcctatct gccattggca aggctgggca 4080ctacccacaa
cctacttcaa ggacctctat accgtgagat tacacacata catcaaaatt
4140tgggaaaagt tctaccaagc tgagagctga tcaccccact cttaggtgct
tatctctgta 4200caccagaaac cttaagaagc aaccagtatt gagagactca
tttatgaaag tctaaaactg 4260gatacaacca aaatgtccac caacagttaa
attatgacat gttcacaatt gagctattac 4320ttaataagga gaattaataa
aataaaactt aagagcatag tttaatctca taaacaagat 4380aataagcaaa
acaaaacatt ttttcatcca tgtaagttta aaagcaggta aaatttaaaa
4440ttaagagaga cataagtttt gaggtagcaa gatggaaact ctggggcttg
gggaatgttc 4500tgtctctctg tatgggatgt gaaagttact attgtggaat
tgggatctat gttcttcctg 4560tatatattgt atacttcata ataacttcac
ctaaagaaat atctaatacc cagtgcatac 4620ataaaagagg atacaaggaa
tgaatcatac gtcaaggcca gaaagacaat aaagtagggg 4680atccaggatc
aaatctccca caaccttgag ccttctacta ttctgccttc cagagctcaa
4740agtacaaaac acataattca aacacatgat ccctccttgg ggtctcttcc
ttcatgcatc 4800gaattagaaa tagccatgta taaaatgaga tagaagagac
cttcatcaac aggtcaaaga 4860atataggtaa ttttgtctgg gtatgaagag
cccacgtatc aaaggttaca ttagggaagg 4920aagaggacac taacagtgac
tttcattctc cccctcttcc tggaggcccc tgcatttagt 4980ccctcgtggg
ctcatccact cagcacacat ttactaagca tcttctcagc ctacactctg
5040aaggcagtgc agaataatgt tagtgtccct tcccccagtt aatatgcagt
ccagtttccc 5100tgctccttcc ctttctcagt ccacataagg atgatgggaa
aggacagtca ccaaatagga 5160gagggcaacc ctttgccttc ctacctcttg
agaatgtaca ttattatcca ctttttgaaa 5220cttcttttaa ttgctttttt
ttaatttgtc ttttcaaata gcataacctt gttcatccat 5280ttctgggaac
caaatttatc aatcaacagt gcctctaatc tggctattaa tacaaaaatg
5340cctcctcaaa atatatatgt tcgagtctta tctaaaacag aacccacaat
aaaaaagaag 5400aaagaataca tataagcatt tatataattc tgagcaacct
tgtgctttgt gaaaaaaata 5460taatctaatg tcacatgctg tattcttttt
atttaacact ggtgaaatta taccattaga 5520gagaaagagg acagatcact
gatcctagga tctagggatg ttacagataa gaaaacaaat 5580gtgacaaaga
gctgtcacaa ggaggatctt caaggtcaca gaatcactgt cttgatttca
5640gtggtggtta catacattta aatatgtgat aaaatgttgt tgaactatat
tcatatattg 5700taccaatgtc aaatgcttaa ttttggctct atagtataat
tatgcactaa ataactattt 5760ggacaaagaa aatgatgttt acatcaaagg
tgaggccata tttgttagga acataactta 5820aaaaccattt tggataacta
atgaaaagcc attttgtgtg ccttggcata tcatgcctaa 5880gctgtcacca
gatagatcta ataagaccta agcctcagaa gcaagcccct gcccagcaag
5940caggcagcac agataagagc taaacccagg acaggccatg atatgctaat
gaactacctt 6000caaggtggtg ttgctgacct agtgaaccag ccccaagctg
tgagccccaa tagcacaaag 6060ctactgccca aagaaattat acaaaaattg
gaactttggg aatggtgtgc aggatcgctc 6120tgctgtatgc ctggaacaca
gcttctctat gttttgtatt gataccagtc tagaagcttc 6180caaaactttc
tcactgaaga agattcccca tgtgggaccc ctacagactc ttttgcccaa
6240acaactgctt ccctcctggt gtgatatctg ttttgctttt atgttagcat
aatattataa 6300ggaatgtttg tgtgaataaa ccaaacatat tttaaaagca
aatattgtat gcacatccta 6360attgctaaaa agtttacagc taatagtccc
atgctctcca caatactgga tccaaataag 6420tcctaatttc aatgttgggc
atctttacag agagaaagac attaaaaatg aagagacatg 6480cagagagtgc
accatgccat cgtggagaca gactgaagtg acacaactgt tagtcaaaga
6540ggattaagga cttccagaag ccaccaaagg aaggaggtat gaagtggttt
ctccctcaga 6600gtatccagag gagactaaac caaccaacac ctttttgctt
aagacttctt gccttcagga 6660ctgtgagaag gtagcttcct attgttctaa
gccccagtat gtggcatttt gttaaggtag 6720agtcaagaaa ccaataaaat
gcagacagac aaaaggatag ctgagttttc caggcccttc 6780cttcttattt
ttggttttgt tggtggtggt ggtggtggtg gtgatggtgg tggttttgtt
6840tatgttttgt ttggggagtt ttttggggtt tttttgggtt ttgtttttgt
tgttgttttg 6900ggggtttttg ttgttgttgt tgtttgcttt tttgtttttt
gttttttgtt tttttgagac 6960agtgtttctc tgtatagccc tggctgtcct
ggagttcctt ctatctctaa tgtctacatc 7020tcagagggga tcctctaatt
tcaaatgagc agtagctctc catttttagc tcttatttat 7080tcatttattt
acttacttac ttattgtctg tagatgaaag aattttggag tgggaaaggg
7140ttcatgagcc cccagcaact aatgaggagc tacagacaat tgatgtttct
ggggaaagga 7200gactcagttt ctttgagagt atagcttctg atgggtcaac
catgttcctg tggctgatgt 7260cacacccagg agtatgcaga caacagaaac
tggagttaat gagttgtttt aaaaataaaa 7320aagggcatga agcttgggat
agaaattaag gataaataca attaaataca ggaaattctg 7380aaagaattaa
taaaaacatt tcttttttta aaaaaaaatc cagaattagc tatgcttctt
7440caaaattgct tctggagaac tttacaagtt aaataagtta tattgtagaa
aaggtagaga 7500ggagaatagt ggaagagaga gataaggaga cttcaaaagg
agtggaggga gatagaggag 7560gagaaagcag aagcaatggc tgatagacac
aggataagag ggaacagaaa ggagaaagag 7620gaagccagga tgggtatttc
tttgcctatc tgtgacttgc acatggtctt ggcaattatt 7680gatgagttca
aggcttaatt cttcacttgt gccaactcaa cagagtcttt ctttcttata
7740accaggcccc cagtatgctc atgtatgtat caggtcctct tatctcctta
tagcaatcct 7800gtttataact gggtaacttt gtgaagggaa ggaagtgcac
actgagatgt gctacaactt 7860tttaatacaa aattttgaag agtttgtaca
atgtatgtat aattaataat taatattatg 7920cactttagat tttgatttca
actcaagata ctaattctat atatatgggt taaatcaata 7980tattaataag
tttaatttca catgcttatt tttattgtgg ttttcgagac agggtttctc
8040tgtatagccc tggctgtcct ggaacccact ttgtagacca ggctggcctc
aaactcagaa 8100acctacctgc ctctgcctct gcctctgcct ctgcctctgc
ctctgcctct gcctctgcct 8160ctgcctctgc ctctgcctct gcctctgcct
ctgcctctgc ctctgcctct gcctctgcct 8220ctgcctctgc ctctgcctct
gcctctgcct ctgcctctgc ctctgcctct gcctctgcct 8280ctgcctagtg
ctggaattaa aggtttgcgc caccacgccc ggtgaaattt ttaaacttta
8340tatatgtctc attctatttc tatcagatag gactgtgtag actgtgctaa
actaataaat 8400gtgccctcaa aagtaatcgc aagttgtatt gttgttgttt
tgctttgctt tgctttgctt 8460tgctttgctt tgctttgctt tgctttgctt
tgctttgctt tgctttgctt tgctttgctt 8520tgctttgctt tgctttgctt
tgcttttttg ttttgggttt ttttccgggg gagggagggt 8580ggagaaagaa
tcttactatg aagctctgac tgtcctggga actcactata tagatcaggc
8640ttgattcaac tcatagagat ctgccttctt ctgcctccca agtgctggga
ataaaggcat 8700acacctccat gcccagatag tgatcccaag ttttagcaaa
agtttctaga cttgacatta 8760atcgatggag atagacatga attacacaaa
gaactaatgt ggagtttacc tgaatcatac 8820tctatacttt atcagagatt
aaattaacat ttaataatcc agtgccaggc tagaggcacc 8880attcaatggc
agtgtttgcc atcatgcata ggcttagtct tcagtgctga aaggcattgg
8940gggcaatatt actcattata cagatgagaa actgggaaag acttgcctca
gattctctac 9000tgaaaggctg agtttgtggc ttctagaaaa tcttttactt
tcaatatttt taatgtataa 9060tttttttatt tccactgatt ttatttttta
tttttaacat ttataagaaa taaatgcaat 9120aaaccaaata catggacaaa
aaaatacaag aatcatatga tcacctcaat ggaaggaaaa 9180aaaaagaaag
aaaaagtctt tgataagatt caacattcat tcttttttta ttagatattt
9240tcttcattta catttcaaat gctatcccca aagcccccta taccttcccc
tgccctgctc 9300cccaacccac ccactcctgc tttctggccc tggcattcct
ctgtactgag gcatatgatc 9360ttcaaaaaac caagggcctc tcctctcatt
ggtggccgac tattaggcca tcttttgcta 9420catatgcaac tagagacaca
gctctggggg ttactggtta gttcatattg ttagtcctcc 9480tatagagttg
cagacccctt tagctccttg gatactttct ctagttcctt cattaggggc
9540cctgtgtccc atccaataga tgactgtgag catccacttc tgtatttgcc
aggcactggc 9600atagcctcac gagaaagaga gagctatgtc aggatcctgt
cagtaaaatc tttctggcat 9660atgcaatagt atctgggttt ggtggttgta
tatgggatgg atccccaagt ggagcagtct 9720ctgaatggtc cttccttcca
tctcagctcc aaactttgtc tctataactc cttccatggg 9780tattttgttc
cccattctaa gaaggagtga agaatccaca ctttggtctt ccttcttctt
9840gagtttcata tgttgcatct tggatattct aagtttctgg gttaatatcc
acgtatcagt 9900gagtgcatat catgcgtgtt attttgtgat tagtttacct
cactcaggat gatatcctcc 9960agatgcatcc atttgcctaa gaatttcatt
aattcactgt ttttaattgc tgaatagtac 10020tccattgtgt aaatgtacca
cattttctgt atccattcct ctgttgaggg gcatctgggt 10080tctttccagc
ttctggctat tataaataag gctgctatga gcatagcgga gcatgtgtcc
10140ttatcaagtt ggaacatctt ctaggtatat gcccaggaga ggaattgctg
gatcttccgg 10200tagtaccatc aacatgcatt cttaataaaa gccctagaac
aaggaggact gtaggaaaca 10260tattccaaca taataaaggt tatgtatgac
aaactcatga ccaatatcat cctaaatgaa 10320tgaaaccatt aataagctcc
attaaaatca gaggactgcc cactatccct acttctcatc 10380cataatgaga
ttgaagcatt agctggagca ataaggcaag agaagggata caaatgggaa
10440aatattaagt caaattgttt tcaattgaag attatattat cttataccca
atgacctcaa 10500attttgacta gaaaaattgt agaaattatc aataatttca
gcaaagtgtt atgatgcacc 10560acatccttat tcttctcccc agcttctgct
tgcttctctc ttcttgctct tcatcctttc 10620tgtccttcca tctgcctgca
ctcttgtctc aagactgagt gcagcgtgta actctcctgt 10680gactgagtat
ctcacaaaac gttctacctg ccaaacctgg atgagccctt tgtctttctg
10740aagctatgag gctctctaca tagactcaag aaggaaatga cagggaggag
gtaataatga 10800agtggggaag gctgacatta gcattgctcc tgtgtggctc
cttaatttct catacttcac 10860actgagatgt tattaactgt gactcatagg
tgaagaagcc agagctaagg ttctcatatt 10920tgagtgttat agaatgagta
gagcagtagt tctcaaacta tgggtcatga ctcctttatg 10980ggtcaaacta
ccctttcaca caggttgcat atcagatatc ctaattttat atacatatat
11040atatgcatat gtatatatat atatttcaca acagtaggaa aattatttag
taatcatttt 11100atagttgtgg gtcatggcaa catgaggaac tgtattaaag
ggttgcagca ttaggaatgt 11160tgagacccac tgtaatagag aatgaggctt
aaggcagggc tataaagccc aatggaccat 11220gtgccttttc caacatttgc
cacatggtaa gctctgtata gactttttaa agaacattgg 11280tttgtaattt
taaatggata agggtcttca ctgtctatca cccatctata taataaatac
11340ataagttttg attccaccat ggattcaaat gcaaaaatcc tcaacctaag
acatagcagt 11400gaaacattga tgaccaaata ggaaatccat gtagagacct
tctatcttct gatggctcca 11460caggcaccat cttgcaacag agttctactt
tgctaccagt aatgaataca gtgtctcaac 11520tcctgccatt gaatcttcag
gaagcccctg aaatgacttg tactacacca tttcttaaag 11580acagaaaagc
taagacttag agggaataaa tgtcatgcct gagatcatgc aaccaattaa
11640gtccaacttg gcctgatcaa gaggcacaat tcaaaagcaa tgttgttcct
tcactagctc 11700ttgtgtatgg ttgctgattc cggaagcaaa gtatcagtga
atatccctag tgggaaaaga 11760cttggaaatc aaatgtctca tttaacagat
taggagatga aacggtagac tctgtgtagt 11820tgtacacccc tgtgatccca
tcgctaggaa gactgaggca ggaagtcctc gagctcaaac 11880cagcttaggc
tacacagaga aactatctaa aaaataatta ctaactactt aataggagat
11940tggatgttaa gatctggtca ctaagaggca gaattgagat tcgaagccag
tattttctac 12000ctggtatgtt ttaaattgca gtaaggatct aagtgtagat
atataataat aagattctat 12060tgatctctgc aacaacagag agtgttagat
ttgtttggaa aaaaatatta tcagccaaca 12120tcttctacca tttcagtata
gcacagagta cccacccata tctccccacc catcccccat 12180accagactgg
ttattgattt tcatggtgac tggcctgaga agattaaaaa aagtaatgct
12240accttattgg gagtgtccca tggaccaaga tagcaactgt catagctacc
gtcacactgc 12300tttgatcaag aagacccttt gaggaactga aaacagaacc
ttaggcacat ctgttgcttt 12360cgctcccatc ctcctccaac agcctgggtg
gtgcactcca caccctttca agtttccaaa 12420gcctcataca cctgctccct
accccagcac ctggccaagg ctgtatccag cactgggatg 12480aaaatgatac
cccacctcca tcttgtttga tattactcta tctcaagccc caggttagtc
12540cccagtccca atgcttttgc acagtcaaaa ctcaacttgg aataatcagt
atccttgaag 12600agttctgata tggtcactgg gcccatatac catgtaagac
atgtggaaaa gatgtttcat 12660ggggcccaga cacgttctag aagtacctga
gagtggcaaa aaatagttgt gctaaatagt 12720ttggccatct ttaggctgag
agactaggaa atacagcgat ggactatatc agcattgcag 12780gatagttgtc
agtaaacacc ccacaaccca taacagaagt attctcttct ttctatatcc
12840cttttccatc catgtagatg gctgtcttca tatttgttct agacggccgg cc
12892946425DNAArtificial SequenceVkP-IGKV1-39/J-Ck-delta2
94ggccggccca catgaaacaa tgggaaccat gtgacaatca cagaggtgtt gttactatag
60caaaagggat tgttactctc cacatccctt taagtaactt gaaggcctga tagacccacc
120ctctaagact tcattagaca ttccctacga atggttatac tctcctgtat
actcccaata 180caactctaaa atatattatt ccatatagtc cttaggtttg
tattaaagtt tgactttttt 240ccttcaaaat atctcttgtc acaacagcgg
ctctagagag aaatacattc cctccaggca 300aatctatgct gcgctggtct
gacctgggac cctggggaca ttgcccctgt gctgagttac 360taagatgagc
cagccctgca gctgtgctca gcctgcccca tgccctgctg attgatttgc
420atgttccaga gcacagcccc ctgccctgaa gactttttta tgggctggtc
gcaccctgtg 480caggagtcag tctcagtcag gagccaccat ggacatgaga
gtgcccgccc agctcctggg 540gctcctgcta ctctggctcc gaggtaagga
tggagaacac taggaattta ctcagccagt 600gtgctcagta ctgactggaa
cttcagggaa gttctctgat aacatgatta atagtaagaa 660tatttgtttt
tatgtttcca atctcaggtg ccagatgtga catccagatg acccagagcc
720ccagcagcct gagcgccagc gtgggcgaca gagtgaccat cacctgcaga
gccagccaga 780gcatcagcag ctacctgaac tggtatcagc agaagcccgg
caaggccccc aagctgctga 840tctacgccgc cagctccctg cagagcggcg
tgcccagcag attcagcggc agcggctccg 900gcaccgactt caccctgacc
atcagcagcc tgcagcccga ggacttcgcc acctactact 960gccagcagag
ctacagcacc ccccccacct tcggccaggg caccaaggtg gagatcaaac
1020gtaagtacac ttttctcatc tttttttatg tgtaagacac aggttttcat
gttaggagtt 1080aaagtcagtt cagaaaatct tgagaaaatg gagagggctc
attatcagtt gacgtggcat 1140acagtgtcag attttctgtt tatcaagcta
gtgagattag gggcaaaaag aggctttagt 1200tgagaggaaa gtaattaata
ctatggtcac catccaagag attggatcgg agaataagca 1260tgagtagtta
ttgagatctg ggtctgactg caggtagcgt ggtcttctag acgtttaagt
1320gggagatttg gaggggatga ggaatgaagg aacttcagga tagaaaaggg
ctgaagtcaa 1380gttcagctcc taaaatggat gtgggagcaa actttgaaga
taaactgaat gacccagagg 1440atgaaacagc gcagatcaaa gaggggcctg
gagctctgag aagagaagga gactcatccg 1500tgttgagttt ccacaagtac
tgtcttgagt tttgcaataa aagtgggata gcagagttga 1560gtgagccgta
ggctgagttc tctcttttgt
ctcctaagtt tttatgacta caaaaatcag 1620tagtatgtcc tgaaataatc
attaagctgt ttgaaagtat gactgcttgc catgtagata 1680ccatggcttg
ctgaataatc agaagaggtg tgactcttat tctaaaattt gtcacaaaat
1740gtcaaaatga gagactctgt aggaacgagt ccttgacaga cagctcaagg
ggtttttttc 1800ctttgtctca tttctacatg aaagtaaatt tgaaatgatc
ttttttatta taagagtaga 1860aatacagttg ggtttgaact atatgtttta
atggccacgg ttttgtaaga catttggtcc 1920tttgttttcc cagttattac
tcgattgtaa ttttatatcg ccagcaatgg actgaaacgg 1980tccgcaacct
cttctttaca actgggtgac ctcgcggctg tgccagccat ttggcgttca
2040ccctgccgct aagggccatg tgaacccccg cggtagcatc ccttgctccg
cgtggaccac 2100tttcctgagg cacagtgata ggaacagagc cactaatctg
aagagaacag agatgtgaca 2160gactacacta atgtgagaaa aacaaggaaa
gggtgactta ttggagattt cagaaataaa 2220atgcatttat tattatattc
ccttatttta attttctatt agggaattag aaagggcata 2280aactgcttta
tccagtgtta tattaaaagc ttaatgtata taatctttta gaggtaaaat
2340ctacagccag caaaagtcat ggtaaatatt ctttgactga actctcacta
aactcctcta 2400aattatatgt catattaact ggttaaatta atataaattt
gtgacatgac cttaactggt 2460taggtaggat atttttcttc atgcaaaaat
atgactaata ataatttagc acaaaaatat 2520ttcccaatac tttaattctg
tgatagaaaa atgtttaact cagctactat aatcccataa 2580ttttgaaaac
tatttatttg gctacaccaa aggaagccat acagaggcta atatcagagt
2640attcttggaa gagacaggag aaaatgaaag ccagtttctg ctcttacctt
atgtgcttgt 2700gttcagactc ccaaacatca ggagtgtcag ataaactggt
ctgaatctct gtctgaagca 2760tggaactgaa aagaatgtag tttcagggaa
gaaaggcaat agaaggaagc ctgagaatat 2820cttcaaaggg tcagactcaa
tttactttct aaagaagtag ctaggaacta gggaataact 2880tagaaacaac
aagattgtat atatgtgcat cctggcccca ttgttcctta tctgtaggga
2940taagcgtgct tttttgtgtg tctgtatata acataactgt ttacacataa
tacactgaaa 3000tggagccctt ccttgttact tcataccatc ctctgtgctt
ccttcctcag gggccgacgc 3060cgctcccacc gtgtccatct tcccccccag
catggaacag ctgacctctg gcggagccac 3120cgtggtctgc ttcgtgaaca
acttctaccc cagagacatc agcgtgaagt ggaagatcga 3180cggcagcgag
cagagggacg gcgtgctgga cagcgtgacc gaccaggaca gcaaggactc
3240cacctacagc atgagcagca ccctgagcct gaccaaggtg gagtacgaga
ggcacaacct 3300gtacacctgc gaggtggtgc acaagaccag ctccagcccc
gtggtcaagt ccttcaaccg 3360gaacgagtgt tgaagacaaa ggtcctgaga
cgccaccacc agctccccag ctccatccta 3420tcttcccttc taaggtcttg
gaggcttccc cacaagcgac ctaccactgt tgcggtgctc 3480caaacctcct
ccccacctcc ttctcctcct cctccctttc cttggctttt atcatgctaa
3540tatttgcaga aaatattcaa taaagtgagt ctttgcactt gagatctctg
tctttcttac 3600taaatggtag taatcagttg tttttccagt tacctgggtt
tctcttctaa agaagttaaa 3660tgtttagttg ccctgaaatc caccacactt
aaaggataaa taaaaccctc cacttgccct 3720ggttggctgt ccactacatg
gcagtccttt ctaaggttca cgagtactat tcatggctta 3780tttctctggg
ccatggtagg tttgaggagg catacttcct agttttcttc ccctaagtcg
3840tcaaagtcct gaagggggac agtctttaca agcacatgtt ctgtaatctg
attcaaccta 3900cccagtaaac ttggcgaagc aaagtagaat cattatcaca
ggaagcaaag gcaacctaaa 3960tgtgcaagca ataggaaaat gtggaagccc
atcatagtac ttggacttca tctgcttttg 4020tgccttcact aagtttttaa
acatgagctg gctcctatct gccattggca aggctgggca 4080ctacccacaa
cctacttcaa ggacctctat accgtgagat tacacacata catcaaaatt
4140tgggaaaagt tctaccaagc tgagagctga tcaccccact cttaggtgct
tatctctgta 4200caccagaaac cttaagaagc aaccagtatt gagagactca
tttatgaaag tctaaaactg 4260gatacaacca aaatgtccac caacagttaa
attatgacat gttcacaatt gagctattac 4320ttaataagga gaattaataa
aataaaactt aagagcatag tttaatctca taaacaagat 4380aataagcaaa
acaaaacatt ttttcatcca tgtaagttta aaagcaggta aaatttaaaa
4440ttaagagaga cataagtttt gaggtagcaa gatggaaact ctggggcttg
gggaatgttc 4500tgtctctctg tatgggatgt gaaagttact attgtggaat
tgggatctat gttcttcctg 4560tatatattgt atacttcata ataacttcac
ctaaagaaat atctaatacc cagtgcatac 4620ataaaagagg atacaaggaa
tgaatcatac gtcaaggcca gaaagacaat aaagtagggg 4680atccaggatc
aaatctccca caaccttgag ccttctacta ttctgccttc cagagctcaa
4740agtacaaaac acataattca aacacatgat ccctccttgg ggtctcttcc
ttcatgcatc 4800gaattagaaa tagccatgta taaaatgaga tagaagagac
cttcatcaac aggtcaaaga 4860atataggtaa ttttgtctgg gtatgaagag
cccacgtatc aaaggttaca ttagggaagg 4920aagaggacac taacagtgac
tttcattctc cccctcttcc tggaggcccc tgcatttagt 4980ccctcgtggg
ctcatccact cagcacacat ttactaagca tcttctcagc ctacactctg
5040aaggcagtgc agaataatgt tagtgtccct tcccccagtt aatatgcagt
ccagtttccc 5100tgctccttcc ctttctcagt ccacataagg atgatgggaa
aggacagtca ccaaatagga 5160gagggcaacc ctttgccttc ctacctcttg
agaatgtaca ttattatcca ctttttgaaa 5220cttcttttaa ttgctttttt
ttaatttgtc ttttcaaata gcataacctt gttcatccat 5280ttctgggaac
caaatttatc aatcaacagt gcctctaatc tggctattaa tacaaaaatg
5340cctcctcaaa atatatatgt tcgagtctta tctaaaacag aacccacaat
aaaaaagaag 5400aaagaataca tataagcatt tatataattc tgagcaacct
tgtgctttgt gaaaaaaata 5460taatctaatg tcacatgctg tattcttttt
atttaacact ggtgaaatta taccattaga 5520gagaaagagg acagatcact
gatcctagga tctagggatg ttacagataa gaaaacaaat 5580gtgacaaaga
gctgtcacaa ggaggatctt caaggtcaca gaatcactgt cttgatttca
5640gtggtggtta catacattta aatatgtgat aaaatgttgt tgaactatat
tcatatattg 5700taccaatgtc aaatgcttaa ttttggctct atagtataat
tatgcactaa ataactattt 5760ggacaaagaa aatgatgttt acatcaaagg
tgaggccata tttgttagga acataactta 5820aaaaccattt tggataacta
atgaaaagcc attttgtgtg ccttggcata tcatgcctaa 5880gctgtcacca
gatagatcta ataagaccta agcctcagaa gcaagcccct gcccagcaag
5940caggcagcac agataagagc taaacccagg acaggccatg atatgctaat
gaactacctt 6000caaggtggtg ttgctgacct agtgaaccag ccccaagctg
tgagccccaa tagcacaaag 6060ctactgccca aagaaattat acaaaaattg
gaactttggg aatggtgtgc aggatcgctc 6120tgctgtatgc ctggaacaca
gcttctctat gttttgtatt gataccagtc tagaagcttc 6180caaaactttc
tcactgaaga agattcccca tgtgggaccc ctacagactc ttttgcccaa
6240acaactgctt ccctcctggt gtgatcatgg accaagatag caactgtcat
agctaccgtc 6300acactgcttt gatcaagaag accctttgag gaactgaaaa
cagaacctta ggcacatctg 6360ttgctttcgc tcccatcctc ctccaacagc
atggctgtct tcatatttgt tctagacggc 6420cggcc 64259513382DNAArtificial
SequenceVkP-IGLV2-14/J-Ck 95ggccggccca catgaaacaa tgggaaccat
gtgacaatca cagaggtgtt gttactatag 60caaaagggat tgttactctc cacatccctt
taagtaactt gaaggcctga tagacccacc 120ctctaagact tcattagaca
ttccctacga atggttatac tctcctgtat actcccaata 180caactctaaa
atatattatt ccatatagtc cttaggtttg tattaaagtt tgactttttt
240ccttcaaaat atctcttgtc acaacagcgg ctctagagag aaatacattc
cctccaggca 300aatctatgct gcgctggtct gacctgggac cctggggaca
ttgcccctgt gctgagttac 360taagatgagc cagccctgca gctgtgctca
gcctgcccca tgccctgctg attgatttgc 420atgttccaga gcacagcccc
ctgccctgaa gactttttta tgggctggtc gcaccctgtg 480caggagtcag
tctcagtcag gagccaccat ggacatgaga gtgcccgccc agctcctggg
540gctcctgcta ctctggctcc gaggtaagga tggagaacac taggaattta
ctcagccagt 600gtgctcagta ctgactggaa cttcagggaa gttctctgat
aacatgatta atagtaagaa 660tatttgtttt tatgtttcca atctcaggtg
ccagatgtca gtctgccctg acccagcccg 720cctctgtgtc tggcagccct
ggccagagca tcaccatcag ctgcaccggc accagcagcg 780acgtgggcgg
ctacaactac gtgtcctggt atcagcagca ccccggcaag gcccccaagc
840tgatgatcta cgaggtgtcc aacagaccca gcggcgtgag caacagattc
agcggcagca 900agagcggcaa caccgccagc ctgaccatca gcggcctcca
ggctgaggac gaggccgact 960actactgcag cagctacacc agcagctcca
ccctggtgtt tggcggcgga acaaagctga 1020ccgtgctgcg taagtacact
tttctcatct ttttttatgt gtaagacaca ggttttcatg 1080ttaggagtta
aagtcagttc agaaaatctt gagaaaatgg agagggctca ttatcagttg
1140acgtggcata cagtgtcaga ttttctgttt atcaagctag tgagattagg
ggcaaaaaga 1200ggctttagtt gagaggaaag taattaatac tatggtcacc
atccaagaga ttggatcgga 1260gaataagcat gagtagttat tgagatctgg
gtctgactgc aggtagcgtg gtcttctaga 1320cgtttaagtg ggagatttgg
aggggatgag gaatgaagga acttcaggat agaaaagggc 1380tgaagtcaag
ttcagctcct aaaatggatg tgggagcaaa ctttgaagat aaactgaatg
1440acccagagga tgaaacagcg cagatcaaag aggggcctgg agctctgaga
agagaaggag 1500actcatccgt gttgagtttc cacaagtact gtcttgagtt
ttgcaataaa agtgggatag 1560cagagttgag tgagccgtag gctgagttct
ctcttttgtc tcctaagttt ttatgactac 1620aaaaatcagt agtatgtcct
gaaataatca ttaagctgtt tgaaagtatg actgcttgcc 1680atgtagatac
catggcttgc tgaataatca gaagaggtgt gactcttatt ctaaaatttg
1740tcacaaaatg tcaaaatgag agactctgta ggaacgagtc cttgacagac
agctcaaggg 1800gtttttttcc tttgtctcat ttctacatga aagtaaattt
gaaatgatct tttttattat 1860aagagtagaa atacagttgg gtttgaacta
tatgttttaa tggccacggt tttgtaagac 1920atttggtcct ttgttttccc
agttattact cgattgtaat tttatatcgc cagcaatgga 1980ctgaaacggt
ccgcaacctc ttctttacaa ctgggtgacc tcgcggctgt gccagccatt
2040tggcgttcac cctgccgcta agggccatgt gaacccccgc ggtagcatcc
cttgctccgc 2100gtggaccact ttcctgaggc acagtgatag gaacagagcc
actaatctga agagaacaga 2160gatgtgacag actacactaa tgtgagaaaa
acaaggaaag ggtgacttat tggagatttc 2220agaaataaaa tgcatttatt
attatattcc cttattttaa ttttctatta gggaattaga 2280aagggcataa
actgctttat ccagtgttat attaaaagct taatgtatat aatcttttag
2340aggtaaaatc tacagccagc aaaagtcatg gtaaatattc tttgactgaa
ctctcactaa 2400actcctctaa attatatgtc atattaactg gttaaattaa
tataaatttg tgacatgacc 2460ttaactggtt aggtaggata tttttcttca
tgcaaaaata tgactaataa taatttagca 2520caaaaatatt tcccaatact
ttaattctgt gatagaaaaa tgtttaactc agctactata 2580atcccataat
tttgaaaact atttattagc ttttgtgttt gacccttccc tagccaaagg
2640caactattta aggacccttt aaaactcttg aaactacttt agagtcatta
agttatttaa 2700ccacttttaa ttactttaaa atgatgtcaa ttccctttta
actattaatt tattttaagg 2760ggggaaaggc tgctcataat tctattgttt
ttcttggtaa agaactctca gttttcgttt 2820ttactacctc tgtcacccaa
gagttggcat ctcaacagag gggactttcc gagaggccat 2880ctggcagttg
cttaagatca gaagtgaagt ctgccagttc ctcccaggca ggtggcccag
2940attacagttg acctgttctg gtgtggctaa aaattgtccc atgtggttac
aaaccattag 3000accagggtct gatgaattgc tcagaatatt tctggacacc
caaatacaga ccctggctta 3060aggccctgtc catacagtag gtttagcttg
gctacaccaa aggaagccat acagaggcta 3120atatcagagt attcttggaa
gagacaggag aaaatgaaag ccagtttctg ctcttacctt 3180atgtgcttgt
gttcagactc ccaaacatca ggagtgtcag ataaactggt ctgaatctct
3240gtctgaagca tggaactgaa aagaatgtag tttcagggaa gaaaggcaat
agaaggaagc 3300ctgagaatat cttcaaaggg tcagactcaa tttactttct
aaagaagtag ctaggaacta 3360gggaataact tagaaacaac aagattgtat
atatgtgcat cctggcccca ttgttcctta 3420tctgtaggga taagcgtgct
tttttgtgtg tctgtatata acataactgt ttacacataa 3480tacactgaaa
tggagccctt ccttgttact tcataccatc ctctgtgctt ccttcctcag
3540gggccgacgc cgctcccacc gtgtccatct tcccccccag catggaacag
ctgacctctg 3600gcggagccac cgtggtctgc ttcgtgaaca acttctaccc
cagagacatc agcgtgaagt 3660ggaagatcga cggcagcgag cagagggacg
gcgtgctgga cagcgtgacc gaccaggaca 3720gcaaggactc cacctacagc
atgagcagca ccctgagcct gaccaaggtg gagtacgaga 3780ggcacaacct
gtacacctgc gaggtggtgc acaagaccag ctccagcccc gtggtcaagt
3840ccttcaaccg gaacgagtgt tgaagacaaa ggtcctgaga cgccaccacc
agctccccag 3900ctccatccta tcttcccttc taaggtcttg gaggcttccc
cacaagcgac ctaccactgt 3960tgcggtgctc caaacctcct ccccacctcc
ttctcctcct cctccctttc cttggctttt 4020atcatgctaa tatttgcaga
aaatattcaa taaagtgagt ctttgcactt gagatctctg 4080tctttcttac
taaatggtag taatcagttg tttttccagt tacctgggtt tctcttctaa
4140agaagttaaa tgtttagttg ccctgaaatc caccacactt aaaggataaa
taaaaccctc 4200cacttgccct ggttggctgt ccactacatg gcagtccttt
ctaaggttca cgagtactat 4260tcatggctta tttctctggg ccatggtagg
tttgaggagg catacttcct agttttcttc 4320ccctaagtcg tcaaagtcct
gaagggggac agtctttaca agcacatgtt ctgtaatctg 4380attcaaccta
cccagtaaac ttggcgaagc aaagtagaat cattatcaca ggaagcaaag
4440gcaacctaaa tgtgcaagca ataggaaaat gtggaagccc atcatagtac
ttggacttca 4500tctgcttttg tgccttcact aagtttttaa acatgagctg
gctcctatct gccattggca 4560aggctgggca ctacccacaa cctacttcaa
ggacctctat accgtgagat tacacacata 4620catcaaaatt tgggaaaagt
tctaccaagc tgagagctga tcaccccact cttaggtgct 4680tatctctgta
caccagaaac cttaagaagc aaccagtatt gagagactca tttatgaaag
4740tctaaaactg gatacaacca aaatgtccac caacagttaa attatgacat
gttcacaatt 4800gagctattac ttaataagga gaattaataa aataaaactt
aagagcatag tttaatctca 4860taaacaagat aataagcaaa acaaaacatt
ttttcatcca tgtaagttta aaagcaggta 4920aaatttaaaa ttaagagaga
cataagtttt gaggtagcaa gatggaaact ctggggcttg 4980gggaatgttc
tgtctctctg tatgggatgt gaaagttact attgtggaat tgggatctat
5040gttcttcctg tatatattgt atacttcata ataacttcac ctaaagaaat
atctaatacc 5100cagtgcatac ataaaagagg atacaaggaa tgaatcatac
gtcaaggcca gaaagacaat 5160aaagtagggg atccaggatc aaatctccca
caaccttgag ccttctacta ttctgccttc 5220cagagctcaa agtacaaaac
acataattca aacacatgat ccctccttgg ggtctcttcc 5280ttcatgcatc
gaattagaaa tagccatgta taaaatgaga tagaagagac cttcatcaac
5340aggtcaaaga atataggtaa ttttgtctgg gtatgaagag cccacgtatc
aaaggttaca 5400ttagggaagg aagaggacac taacagtgac tttcattctc
cccctcttcc tggaggcccc 5460tgcatttagt ccctcgtggg ctcatccact
cagcacacat ttactaagca tcttctcagc 5520ctacactctg aaggcagtgc
agaataatgt tagtgtccct tcccccagtt aatatgcagt 5580ccagtttccc
tgctccttcc ctttctcagt ccacataagg atgatgggaa aggacagtca
5640ccaaatagga gagggcaacc ctttgccttc ctacctcttg agaatgtaca
ttattatcca 5700ctttttgaaa cttcttttaa ttgctttttt ttaatttgtc
ttttcaaata gcataacctt 5760gttcatccat ttctgggaac caaatttatc
aatcaacagt gcctctaatc tggctattaa 5820tacaaaaatg cctcctcaaa
atatatatgt tcgagtctta tctaaaacag aacccacaat 5880aaaaaagaag
aaagaataca tataagcatt tatataattc tgagcaacct tgtgctttgt
5940gaaaaaaata taatctaatg tcacatgctg tattcttttt atttaacact
ggtgaaatta 6000taccattaga gagaaagagg acagatcact gatcctagga
tctagggatg ttacagataa 6060gaaaacaaat gtgacaaaga gctgtcacaa
ggaggatctt caaggtcaca gaatcactgt 6120cttgatttca gtggtggtta
catacattta aatatgtgat aaaatgttgt tgaactatat 6180tcatatattg
taccaatgtc aaatgcttaa ttttggctct atagtataat tatgcactaa
6240ataactattt ggacaaagaa aatgatgttt acatcaaagg tgaggccata
tttgttagga 6300acataactta aaaaccattt tggataacta atgaaaagcc
attttgtgtg ccttggcata 6360tcatgcctaa gctgtcacca gatagatcta
ataagaccta agcctcagaa gcaagcccct 6420gcccagcaag caggcagcac
agataagagc taaacccagg acaggccatg atatgctaat 6480gaactacctt
caaggtggtg ttgctgacct agtgaaccag ccccaagctg tgagccccaa
6540tagcacaaag ctactgccca aagaaattat acaaaaattg gaactttggg
aatggtgtgc 6600aggatcgctc tgctgtatgc ctggaacaca gcttctctat
gttttgtatt gataccagtc 6660tagaagcttc caaaactttc tcactgaaga
agattcccca tgtgggaccc ctacagactc 6720ttttgcccaa acaactgctt
ccctcctggt gtgatatctg ttttgctttt atgttagcat 6780aatattataa
ggaatgtttg tgtgaataaa ccaaacatat tttaaaagca aatattgtat
6840gcacatccta attgctaaaa agtttacagc taatagtccc atgctctcca
caatactgga 6900tccaaataag tcctaatttc aatgttgggc atctttacag
agagaaagac attaaaaatg 6960aagagacatg cagagagtgc accatgccat
cgtggagaca gactgaagtg acacaactgt 7020tagtcaaaga ggattaagga
cttccagaag ccaccaaagg aaggaggtat gaagtggttt 7080ctccctcaga
gtatccagag gagactaaac caaccaacac ctttttgctt aagacttctt
7140gccttcagga ctgtgagaag gtagcttcct attgttctaa gccccagtat
gtggcatttt 7200gttaaggtag agtcaagaaa ccaataaaat gcagacagac
aaaaggatag ctgagttttc 7260caggcccttc cttcttattt ttggttttgt
tggtggtggt ggtggtggtg gtgatggtgg 7320tggttttgtt tatgttttgt
ttggggagtt ttttggggtt tttttgggtt ttgtttttgt 7380tgttgttttg
ggggtttttg ttgttgttgt tgtttgcttt tttgtttttt gttttttgtt
7440tttttgagac agtgtttctc tgtatagccc tggctgtcct ggagttcctt
ctatctctaa 7500tgtctacatc tcagagggga tcctctaatt tcaaatgagc
agtagctctc catttttagc 7560tcttatttat tcatttattt acttacttac
ttattgtctg tagatgaaag aattttggag 7620tgggaaaggg ttcatgagcc
cccagcaact aatgaggagc tacagacaat tgatgtttct 7680ggggaaagga
gactcagttt ctttgagagt atagcttctg atgggtcaac catgttcctg
7740tggctgatgt cacacccagg agtatgcaga caacagaaac tggagttaat
gagttgtttt 7800aaaaataaaa aagggcatga agcttgggat agaaattaag
gataaataca attaaataca 7860ggaaattctg aaagaattaa taaaaacatt
tcttttttta aaaaaaaatc cagaattagc 7920tatgcttctt caaaattgct
tctggagaac tttacaagtt aaataagtta tattgtagaa 7980aaggtagaga
ggagaatagt ggaagagaga gataaggaga cttcaaaagg agtggaggga
8040gatagaggag gagaaagcag aagcaatggc tgatagacac aggataagag
ggaacagaaa 8100ggagaaagag gaagccagga tgggtatttc tttgcctatc
tgtgacttgc acatggtctt 8160ggcaattatt gatgagttca aggcttaatt
cttcacttgt gccaactcaa cagagtcttt 8220ctttcttata accaggcccc
cagtatgctc atgtatgtat caggtcctct tatctcctta 8280tagcaatcct
gtttataact gggtaacttt gtgaagggaa ggaagtgcac actgagatgt
8340gctacaactt tttaatacaa aattttgaag agtttgtaca atgtatgtat
aattaataat 8400taatattatg cactttagat tttgatttca actcaagata
ctaattctat atatatgggt 8460taaatcaata tattaataag tttaatttca
catgcttatt tttattgtgg ttttcgagac 8520agggtttctc tgtatagccc
tggctgtcct ggaacccact ttgtagacca ggctggcctc 8580aaactcagaa
acctacctgc ctctgcctct gcctctgcct ctgcctctgc ctctgcctct
8640gcctctgcct ctgcctctgc ctctgcctct gcctctgcct ctgcctctgc
ctctgcctct 8700gcctctgcct ctgcctctgc ctctgcctct gcctctgcct
ctgcctctgc ctctgcctct 8760gcctctgcct ctgcctagtg ctggaattaa
aggtttgcgc caccacgccc ggtgaaattt 8820ttaaacttta tatatgtctc
attctatttc tatcagatag gactgtgtag actgtgctaa 8880actaataaat
gtgccctcaa aagtaatcgc aagttgtatt gttgttgttt tgctttgctt
8940tgctttgctt tgctttgctt tgctttgctt tgctttgctt tgctttgctt
tgctttgctt 9000tgctttgctt tgctttgctt tgctttgctt tgcttttttg
ttttgggttt ttttccgggg 9060gagggagggt ggagaaagaa tcttactatg
aagctctgac tgtcctggga actcactata 9120tagatcaggc ttgattcaac
tcatagagat ctgccttctt ctgcctccca agtgctggga 9180ataaaggcat
acacctccat gcccagatag tgatcccaag ttttagcaaa agtttctaga
9240cttgacatta atcgatggag atagacatga attacacaaa gaactaatgt
ggagtttacc 9300tgaatcatac tctatacttt atcagagatt aaattaacat
ttaataatcc agtgccaggc 9360tagaggcacc attcaatggc agtgtttgcc
atcatgcata ggcttagtct tcagtgctga 9420aaggcattgg gggcaatatt
actcattata cagatgagaa actgggaaag acttgcctca 9480gattctctac
tgaaaggctg agtttgtggc ttctagaaaa tcttttactt tcaatatttt
9540taatgtataa tttttttatt tccactgatt ttatttttta tttttaacat
ttataagaaa 9600taaatgcaat aaaccaaata catggacaaa aaaatacaag
aatcatatga tcacctcaat 9660ggaaggaaaa aaaaagaaag aaaaagtctt
tgataagatt caacattcat tcttttttta 9720ttagatattt tcttcattta
catttcaaat gctatcccca aagcccccta taccttcccc 9780tgccctgctc
cccaacccac ccactcctgc tttctggccc tggcattcct ctgtactgag
9840gcatatgatc ttcaaaaaac caagggcctc tcctctcatt ggtggccgac
tattaggcca 9900tcttttgcta catatgcaac tagagacaca gctctggggg
ttactggtta gttcatattg 9960ttagtcctcc tatagagttg cagacccctt
tagctccttg gatactttct ctagttcctt 10020cattaggggc cctgtgtccc
atccaataga tgactgtgag catccacttc tgtatttgcc 10080aggcactggc
atagcctcac gagaaagaga gagctatgtc
aggatcctgt cagtaaaatc 10140tttctggcat atgcaatagt atctgggttt
ggtggttgta tatgggatgg atccccaagt 10200ggagcagtct ctgaatggtc
cttccttcca tctcagctcc aaactttgtc tctataactc 10260cttccatggg
tattttgttc cccattctaa gaaggagtga agaatccaca ctttggtctt
10320ccttcttctt gagtttcata tgttgcatct tggatattct aagtttctgg
gttaatatcc 10380acgtatcagt gagtgcatat catgcgtgtt attttgtgat
tagtttacct cactcaggat 10440gatatcctcc agatgcatcc atttgcctaa
gaatttcatt aattcactgt ttttaattgc 10500tgaatagtac tccattgtgt
aaatgtacca cattttctgt atccattcct ctgttgaggg 10560gcatctgggt
tctttccagc ttctggctat tataaataag gctgctatga gcatagcgga
10620gcatgtgtcc ttatcaagtt ggaacatctt ctaggtatat gcccaggaga
ggaattgctg 10680gatcttccgg tagtaccatc aacatgcatt cttaataaaa
gccctagaac aaggaggact 10740gtaggaaaca tattccaaca taataaaggt
tatgtatgac aaactcatga ccaatatcat 10800cctaaatgaa tgaaaccatt
aataagctcc attaaaatca gaggactgcc cactatccct 10860acttctcatc
cataatgaga ttgaagcatt agctggagca ataaggcaag agaagggata
10920caaatgggaa aatattaagt caaattgttt tcaattgaag attatattat
cttataccca 10980atgacctcaa attttgacta gaaaaattgt agaaattatc
aataatttca gcaaagtgtt 11040atgatgcacc acatccttat tcttctcccc
agcttctgct tgcttctctc ttcttgctct 11100tcatcctttc tgtccttcca
tctgcctgca ctcttgtctc aagactgagt gcagcgtgta 11160actctcctgt
gactgagtat ctcacaaaac gttctacctg ccaaacctgg atgagccctt
11220tgtctttctg aagctatgag gctctctaca tagactcaag aaggaaatga
cagggaggag 11280gtaataatga agtggggaag gctgacatta gcattgctcc
tgtgtggctc cttaatttct 11340catacttcac actgagatgt tattaactgt
gactcatagg tgaagaagcc agagctaagg 11400ttctcatatt tgagtgttat
agaatgagta gagcagtagt tctcaaacta tgggtcatga 11460ctcctttatg
ggtcaaacta ccctttcaca caggttgcat atcagatatc ctaattttat
11520atacatatat atatgcatat gtatatatat atatttcaca acagtaggaa
aattatttag 11580taatcatttt atagttgtgg gtcatggcaa catgaggaac
tgtattaaag ggttgcagca 11640ttaggaatgt tgagacccac tgtaatagag
aatgaggctt aaggcagggc tataaagccc 11700aatggaccat gtgccttttc
caacatttgc cacatggtaa gctctgtata gactttttaa 11760agaacattgg
tttgtaattt taaatggata agggtcttca ctgtctatca cccatctata
11820taataaatac ataagttttg attccaccat ggattcaaat gcaaaaatcc
tcaacctaag 11880acatagcagt gaaacattga tgaccaaata ggaaatccat
gtagagacct tctatcttct 11940gatggctcca caggcaccat cttgcaacag
agttctactt tgctaccagt aatgaataca 12000gtgtctcaac tcctgccatt
gaatcttcag gaagcccctg aaatgacttg tactacacca 12060tttcttaaag
acagaaaagc taagacttag agggaataaa tgtcatgcct gagatcatgc
12120aaccaattaa gtccaacttg gcctgatcaa gaggcacaat tcaaaagcaa
tgttgttcct 12180tcactagctc ttgtgtatgg ttgctgattc cggaagcaaa
gtatcagtga atatccctag 12240tgggaaaaga cttggaaatc aaatgtctca
tttaacagat taggagatga aacggtagac 12300tctgtgtagt tgtacacccc
tgtgatccca tcgctaggaa gactgaggca ggaagtcctc 12360gagctcaaac
cagcttaggc tacacagaga aactatctaa aaaataatta ctaactactt
12420aataggagat tggatgttaa gatctggtca ctaagaggca gaattgagat
tcgaagccag 12480tattttctac ctggtatgtt ttaaattgca gtaaggatct
aagtgtagat atataataat 12540aagattctat tgatctctgc aacaacagag
agtgttagat ttgtttggaa aaaaatatta 12600tcagccaaca tcttctacca
tttcagtata gcacagagta cccacccata tctccccacc 12660catcccccat
accagactgg ttattgattt tcatggtgac tggcctgaga agattaaaaa
12720aagtaatgct accttattgg gagtgtccca tggaccaaga tagcaactgt
catagctacc 12780gtcacactgc tttgatcaag aagacccttt gaggaactga
aaacagaacc ttaggcacat 12840ctgttgcttt cgctcccatc ctcctccaac
agcctgggtg gtgcactcca caccctttca 12900agtttccaaa gcctcataca
cctgctccct accccagcac ctggccaagg ctgtatccag 12960cactgggatg
aaaatgatac cccacctcca tcttgtttga tattactcta tctcaagccc
13020caggttagtc cccagtccca atgcttttgc acagtcaaaa ctcaacttgg
aataatcagt 13080atccttgaag agttctgata tggtcactgg gcccatatac
catgtaagac atgtggaaaa 13140gatgtttcat ggggcccaga cacgttctag
aagtacctga gagtggcaaa aaatagttgt 13200gctaaatagt ttggccatct
ttaggctgag agactaggaa atacagcgat ggactatatc 13260agcattgcag
gatagttgtc agtaaacacc ccacaaccca taacagaagt attctcttct
13320ttctatatcc cttttccatc catgtagatg gctgtcttca tatttgttct
agacggccgg 13380cc 13382964638DNAArtificial
SequencepSELECT-IGKV1-39/J-Ck 96gcggccgcaa taaaatatct ttattttcat
tacatctgtg tgttggtttt ttgtgtgaat 60cgtaactaac atacgctctc catcaaaaca
aaacgaaaca aaacaaacta gcaaaatagg 120ctgtccccag tgcaagtgca
ggtgccagaa catttctcta tcgaaggatc tgcgatcgct 180ccggtgcccg
tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag
240gggtcggcaa ttgaacgggt gcctagagaa ggtggcgcgg ggtaaactgg
gaaagtgatg 300tcgtgtactg gctccgcctt tttcccgagg gtgggggaga
accgtatata agtgcagtag 360tcgccgtgaa cgttcttttt cgcaacgggt
ttgccgccag aacacagctg aagcttcgag 420gggctcgcat ctctccttca
cgcgcccgcc gccctacctg aggccgccat ccacgccggt 480tgagtcgcgt
tctgccgcct cccgcctgtg gtgcctcctg aactgcgtcc gccgtctagg
540taagtttaaa gctcaggtcg agaccgggcc tttgtccggc gctcccttgg
agcctaccta 600gactcagccg gctctccacg ctttgcctga ccctgcttgc
tcaactctac gtctttgttt 660cgttttctgt tctgcgccgt tacagatcca
agctgtgacc ggcgcctacc tgagatcacc 720ggcgtgtcga cgccaccatg
gacatgagag tgcccgccca gctcctgggg ctcctgctac 780tctggctccg
aggtaaggat ggagaacact aggaatttac tcagccagtg tgctcagtac
840tgactggaac ttcagggaag ttctctgata acatgattaa tagtaagaat
atttgttttt 900atgtttccaa tctcaggtgc cagatgtgac atccagatga
cccagagccc cagcagcctg 960agcgccagcg tgggcgacag agtgaccatc
acctgcagag ccagccagag catcagcagc 1020tacctgaact ggtatcagca
gaagcccggc aaggccccca agctgctgat ctacgccgcc 1080agctccctgc
agagcggcgt gcccagcaga ttcagcggca gcggctccgg caccgacttc
1140accctgacca tcagcagcct gcagcccgag gacttcgcca cctactactg
ccagcagagc 1200tacagcaccc cccccacctt cggccagggc accaaggtgg
agatcaagag agccgacgcc 1260gctcccaccg tgtccatctt cccccccagc
atggaacagc tgacctctgg cggagccacc 1320gtggtctgct tcgtgaacaa
cttctacccc agagacatca gcgtgaagtg gaagatcgac 1380ggcagcgagc
agagggacgg cgtgctggac agcgtgaccg accaggacag caaggactcc
1440acctacagca tgagcagcac cctgagcctg accaaggtgg agtacgagag
gcacaacctg 1500tacacctgcg aggtggtgca caagaccagc tccagccccg
tggtcaagtc cttcaaccgg 1560aacgagtgtt gagctagctg gccagacatg
ataagataca ttgatgagtt tggacaaacc 1620acaactagaa tgcagtgaaa
aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta 1680tttgtaacca
ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
1740tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct
ctacaaatgt 1800ggtatggaat tctaaaatac agcatagcaa aactttaacc
tccaaatcaa gcctctactt 1860gaatcctttt ctgagggatg aataaggcat
aggcatcagg ggctgttgcc aatgtgcatt 1920agctgtttgc agcctcacct
tctttcatgg agtttaagat atagtgtatt ttcccaaggt 1980ttgaactagc
tcttcatttc tttatgtttt aaatgcactg acctcccaca ttcccttttt
2040agtaaaatat tcagaaataa tttaaataca tcattgcaat gaaaataaat
gttttttatt 2100aggcagaatc cagatgctca aggcccttca taatatcccc
cagtttagta gttggactta 2160gggaacaaag gaacctttaa tagaaattgg
acagcaagaa agcgagcttc tagcgaattc 2220tcgactcatt cctttgccct
cggacgagtg ctggggcgtc ggtttccact atcggcgagt 2280acttctacac
agccatcggt ccagacggcc gcgcttctgc gggcgatttg tgtacgcccg
2340acagtcccgg ctccggatcg gacgattgcg tcgcatcgac cctgcgccca
agctgcatca 2400tcgaaattgc cgtcaaccaa gctctgatag agttggtcaa
gaccaatgcg gagcatatac 2460gcccggagcc gcggcgatcc tgcaagctcc
ggatgcctcc gctcgaagta gcgcgtctgc 2520tgctccatac aagccaacca
cggcctccag aagaagatgt tggcgacctc gtattgggaa 2580tccccgaaca
tcgcctcgct ccagtcaatg accgctgtta tgcggccatt gtccgtcagg
2640acattgttgg agccgaaatc cgcgtgcacg aggtgccgga cttcggggca
gtcctcggcc 2700caaagcatca gctcatcgag agcctgcgcg acggacgcac
tgacggtgtc gtccatcaca 2760gtttgccagt gatacacatg gggatcagca
atcgcgcata tgaaatcacg ccatgtagtg 2820tattgaccga ttccttgcgg
tccgaatggg ccgaacccgc tcgtctggct aagatcggcc 2880gcagcgatcg
catccatgag ctccgcgacg ggttgcagaa cagcgggcag ttcggtttca
2940ggcaggtctt gcaacgtgac accctgtgca cggcgggaga tgcaataggt
caggctctcg 3000ctgaattccc caatgtcaag cacttccgga atcgggagcg
cggccgatgc aaagtgccga 3060taaacataac gatctttgta gaaaccatcg
gcgcagctat ttacccgcag gacatatcca 3120cgccctccta catcgaagct
gaaagcacga gattcttcgc cctccgagag ctgcatcagg 3180tcggagacgc
tgtcgaactt ttcgatcaga aacttctcga cagacgtcgc ggtgagttca
3240ggctttttca tgatggccct cctatagtga gtcgtattat actatgccga
tatactatgc 3300cgatgattaa ttgtcaaaac agcgtggatg gcgtctccag
cttatctgac ggttcactaa 3360acgagctctg cttatataga cctcccaccg
tacacgccta ccgcccattt gcgtcaatgg 3420ggcggagttg ttacgacatt
ttggaaagtc ccgttgattt actagtcaaa acaaactccc 3480attgacgtca
atggggtgga gacttggaaa tccccgtgag tcaaaccgct atccacgccc
3540attgatgtac tgccaaaacc gcatcatcat ggtaatagcg atgactaata
cgtagatgta 3600ctgccaagta ggaaagtccc ataaggtcat gtactgggca
taatgccagg cgggccattt 3660accgtcattg acgtcaatag ggggcgtact
tggcatatga tacacttgat gtactgccaa 3720gtgggcagtt taccgtaaat
actccaccca ttgacgtcaa tggaaagtcc ctattggcgt 3780tactatggga
acatacgtca ttattgacgt caatgggcgg gggtcgttgg gcggtcagcc
3840aggcgggcca tttaccgtaa gttatgtaac gcctgcaggt taattaagaa
catgtgagca 3900aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
tgctggcgtt tttccatagg 3960ctccgccccc ctgacgagca tcacaaaaat
cgacgctcaa gtcagaggtg gcgaaacccg 4020acaggactat aaagatacca
ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 4080ccgaccctgc
cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
4140tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
caagctgggc 4200tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
tatccggtaa ctatcgtctt 4260gagtccaacc cggtaagaca cgacttatcg
ccactggcag cagccactgg taacaggatt 4320agcagagcga ggtatgtagg
cggtgctaca gagttcttga agtggtggcc taactacggc 4380tacactagaa
gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
4440agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
tttttttgtt 4500tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
aagatccttt gatcttttct 4560acggggtctg acgctcagtg gaacgaaaac
tcacgttaag ggattttggt catggctagt 4620taattaacat ttaaatca
4638975349DNAArtificial SequencepSelect-IGVL2-14/J-Ck 97gcggccgcaa
taaaatatct ttattttcat tacatctgtg tgttggtttt ttgtgtgaat 60cgtaactaac
atacgctctc catcaaaaca aaacgaaaca aaacaaacta gcaaaatagg
120ctgtccccag tgcaagtgca ggtgccagaa catttctcta tcgaaggatc
tgcgatcgct 180ccggtgcccg tcagtgggca gagcgcacat cgcccacagt
ccccgagaag ttggggggag 240gggtcggcaa ttgaacgggt gcctagagaa
ggtggcgcgg ggtaaactgg gaaagtgatg 300tcgtgtactg gctccgcctt
tttcccgagg gtgggggaga accgtatata agtgcagtag 360tcgccgtgaa
cgttcttttt cgcaacgggt ttgccgccag aacacagctg aagcttcgag
420gggctcgcat ctctccttca cgcgcccgcc gccctacctg aggccgccat
ccacgccggt 480tgagtcgcgt tctgccgcct cccgcctgtg gtgcctcctg
aactgcgtcc gccgtctagg 540taagtttaaa gctcaggtcg agaccgggcc
tttgtccggc gctcccttgg agcctaccta 600gactcagccg gctctccacg
ctttgcctga ccctgcttgc tcaactctac gtctttgttt 660cgttttctgt
tctgcgccgt tacagatcca agctgtgacc ggcgcctacc tgagatcacc
720ggcgtgtcga cgccaccatg gacatgagag tgcccgccca gctcctgggg
ctcctgctac 780tctggctccg aggtaaggat ggagaacact aggaatttac
tcagccagtg tgctcagtac 840tgactggaac ttcagggaag ttctctgata
acatgattaa tagtaagaat atttgttttt 900atgtttccaa tctcaggtgc
cagatgtcag tctgccctga cccagcccgc ctctgtgtct 960ggcagccctg
gccagagcat caccatcagc tgcaccggca ccagcagcga cgtgggcggc
1020tacaactacg tgtcctggta tcagcagcac cccggcaagg cccccaagct
gatgatctac 1080gaggtgtcca acagacccag cggcgtgagc aacagattca
gcggcagcaa gagcggcaac 1140accgccagcc tgaccatcag cggcctccag
gctgaggacg aggccgacta ctactgcagc 1200agctacacca gcagctccac
cctggtgttt ggcggcggaa caaagctgac cgtgctgaga 1260gccgacgccg
ctcccaccgt gtccatcttc ccccccagca tggaacagct gacctctggc
1320ggagccaccg tggtctgctt cgtgaacaac ttctacccca gagacatcag
cgtgaagtgg 1380aagatcgacg gcagcgagca gagggacggc gtgctggaca
gcgtgaccga ccaggacagc 1440aaggactcca cctacagcat gagcagcacc
ctgagcctga ccaaggtgga gtacgagagg 1500cacaacctgt acacctgcga
ggtggtgcac aagaccagct ccagccccgt ggtcaagtcc 1560ttcaaccgga
acgagtgttg agctagctgg ccagacatga taagatacat tgatgagttt
1620ggacaaacca caactagact gactcagcct gcctccgtgt ctgggtctcc
tggacagtcg 1680atcaccatct cctgcactgg aaccagcagt gacgttggtg
gttataacta tgtctcctgg 1740taccaacagc acccaggcaa agcccccaaa
ctcatgattt atgaggtcag taatcggccc 1800tcaggggttt ctaatcgctt
ctctggctcc aagtctggca acacggcctc cctgaccatc 1860tctgggctcc
aggctgagga cgaggctgat tattactgca gctcatatac aagcagcagc
1920actctcgtat tcggcggagg gaccaagctg accgtcctac gggctgatgc
tgcaccaact 1980gtatccatct tcccaccatc catggaacag ttaacatctg
gaggtgccac agtcgtgtgc 2040ttcgtgaaca acttctatcc cagagacatc
agtgtcaagt ggaagattga tggcagtgaa 2100caacgagatg gtgtcctgga
cagtgttact gatcaggaca gcaaagacag cacgtacagc 2160atgagcagca
ccctctcgtt gaccaaggtt gaatatgaaa ggcataacct ctatacctgt
2220gaggttgttc ataagacatc atcctcaccc gtcgtcaaga gcttcaacag
gaatgagtgt 2280taggctagct ggccagacat gataagatac attgatgagt
ttggacaaac cacaactaga 2340atgcagtgaa aaaaatgctt tatttgtgaa
atttgtgatg ctattgcttt atttgtaacc 2400attataagct gcaataaaca
agttaacaac aacaattgca ttcattttat gtttcaggtt 2460cagggggagg
tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtatggaa
2520ttctaaaata cagcatagca aaactttaac ctccaaatca agcctctact
tgaatccttt 2580tctgagggat gaataaggca taggcatcag gggctgttgc
caatgtgcat tagctgtttg 2640cagcctcacc ttctttcatg gagtttaaga
tatagtgtat tttcccaagg tttgaactag 2700ctcttcattt ctttatgttt
taaatgcact gacctcccac attccctttt tagtaaaata 2760ttcagaaata
atttaaatac atcattgcaa tgaaaataaa tgttttttat taggcagaat
2820ccagatgctc aaggcccttc ataatatccc ccagtttagt agttggactt
agggaacaaa 2880ggaaccttta atagaaattg gacagcaaga aagcgagctt
ctagcgaatt ctcgactcat 2940tcctttgccc tcggacgagt gctggggcgt
cggtttccac tatcggcgag tacttctaca 3000cagccatcgg tccagacggc
cgcgcttctg cgggcgattt gtgtacgccc gacagtcccg 3060gctccggatc
ggacgattgc gtcgcatcga ccctgcgccc aagctgcatc atcgaaattg
3120ccgtcaacca agctctgata gagttggtca agaccaatgc ggagcatata
cgcccggagc 3180cgcggcgatc ctgcaagctc cggatgcctc cgctcgaagt
agcgcgtctg ctgctccata 3240caagccaacc acggcctcca gaagaagatg
ttggcgacct cgtattggga atccccgaac 3300atcgcctcgc tccagtcaat
gaccgctgtt atgcggccat tgtccgtcag gacattgttg 3360gagccgaaat
ccgcgtgcac gaggtgccgg acttcggggc agtcctcggc ccaaagcatc
3420agctcatcga gagcctgcgc gacggacgca ctgacggtgt cgtccatcac
agtttgccag 3480tgatacacat ggggatcagc aatcgcgcat atgaaatcac
gccatgtagt gtattgaccg 3540attccttgcg gtccgaatgg gccgaacccg
ctcgtctggc taagatcggc cgcagcgatc 3600gcatccatga gctccgcgac
gggttgcaga acagcgggca gttcggtttc aggcaggtct 3660tgcaacgtga
caccctgtgc acggcgggag atgcaatagg tcaggctctc gctgaattcc
3720ccaatgtcaa gcacttccgg aatcgggagc gcggccgatg caaagtgccg
ataaacataa 3780cgatctttgt agaaaccatc ggcgcagcta tttacccgca
ggacatatcc acgccctcct 3840acatcgaagc tgaaagcacg agattcttcg
ccctccgaga gctgcatcag gtcggagacg 3900ctgtcgaact tttcgatcag
aaacttctcg acagacgtcg cggtgagttc aggctttttc 3960atgatggccc
tcctatagtg agtcgtatta tactatgccg atatactatg ccgatgatta
4020attgtcaaaa cagcgtggat ggcgtctcca gcttatctga cggttcacta
aacgagctct 4080gcttatatag acctcccacc gtacacgcct accgcccatt
tgcgtcaatg gggcggagtt 4140gttacgacat tttggaaagt cccgttgatt
tactagtcaa aacaaactcc cattgacgtc 4200aatggggtgg agacttggaa
atccccgtga gtcaaaccgc tatccacgcc cattgatgta 4260ctgccaaaac
cgcatcatca tggtaatagc gatgactaat acgtagatgt actgccaagt
4320aggaaagtcc cataaggtca tgtactgggc ataatgccag gcgggccatt
taccgtcatt 4380gacgtcaata gggggcgtac ttggcatatg atacacttga
tgtactgcca agtgggcagt 4440ttaccgtaaa tactccaccc attgacgtca
atggaaagtc cctattggcg ttactatggg 4500aacatacgtc attattgacg
tcaatgggcg ggggtcgttg ggcggtcagc caggcgggcc 4560atttaccgta
agttatgtaa cgcctgcagg ttaattaaga acatgtgagc aaaaggccag
4620caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
gctccgcccc 4680cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
ggcgaaaccc gacaggacta 4740taaagatacc aggcgtttcc ccctggaagc
tccctcgtgc gctctcctgt tccgaccctg 4800ccgcttaccg gatacctgtc
cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 4860tcacgctgta
ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
4920gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
tgagtccaac 4980ccggtaagac acgacttatc gccactggca gcagccactg
gtaacaggat tagcagagcg 5040aggtatgtag gcggtgctac agagttcttg
aagtggtggc ctaactacgg ctacactaga 5100agaacagtat ttggtatctg
cgctctgctg aagccagtta ccttcggaaa aagagttggt 5160agctcttgat
ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
5220cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
tacggggtct 5280gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
tcatggctag ttaattaaca 5340tttaaatca 5349986772DNAArtificial
SequenceMV1043 98cttgatttgg gtgatggttc acgtagtggg ccatcgccct
gatagacggt ttttcgccct 60ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
tccaaactgg aacaacactc 120aactctatct cgggctattc ttttgattta
taagggattt tgccgatttc ggtctattgg 180ttaaaaaatg agctgattta
acaaaaattt aacgcgaatt ttaacaaaat attaacgttt 240acaattttat
ggtgcagtct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
300cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
ggcatccgct 360tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
agaggttttc accgtcatca 420ccgaaacgcg cgagacgaaa gggcctcgtg
atacgcctat ttttataggt taatgtcatg 480ataataatgg tttcttagac
gtcaggtggc acttttcggg gaaatgtgcg cggaacccct 540atttgtttat
ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
600taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
ccgtgtcgcc 660cttattccct tttttgcggc attttgcctt cctgtttttg
ctcacccaga aacgctggtg 720aaagtaaaag atgctgaaga tcagttgggt
gcccgagtgg gttacatcga actggatctc 780aacagcggta agatccttga
gagttttcgc cccgaagaac gttttccaat gatgagcact 840tttaaagttc
tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
900ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
cacagaaaag 960catcttacgg atggcatgac agtaagagaa ttatgcagtg
ctgccataac catgagtgat 1020aacactgcgg ccaacttact tctgacaacg
atcggaggac cgaaggagct aaccgctttt 1080ttgcacaaca tgggggatca
tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa 1140gccataccaa
acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
1200aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
agactggatg 1260gaggcggata aagttgcagg accacttctg cgctcggccc
ttccggctgg ctggtttatt 1320gctgataaat ctggagccgg tgagcgtggg
tctcgcggta tcattgcagc actggggcca 1380gatggtaagc cctcccgtat
cgtagttatc tacacgacgg ggagtcaggc aactatggat 1440gaacgaaata
gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
1500gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
atttaaaagg 1560atctaggtga agatcctttt tgataatctc atgaccaaaa
tcccttaacg tgagttttcg 1620ttccactgag cgtcagaccc cgtagaaaag
atcaaaggat cttcttgaga tccttttttt 1680ctgcgcgtaa tctgctgctt
gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 1740ccggatcaag
agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
1800ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
ctctgtagca 1860ccgcctacat acctcgctct gctaatcctg ttaccagtgg
ctgctgccag tggcgataag 1920tcgtgtctta ccgggttgga ctcaagacga
tagttaccgg ataaggcgca gcggtcgggc 1980tgaacggggg gttcgtgcat
acagcccagc ttggagcgaa cgacctacac cgaactgaga 2040tacctacagc
gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
2100tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
agggggaaac 2160gcctggtatc tttatagtcc tgtcgggttt cgccacctct
gacttgagcg tcgatttttg 2220tgatgctcgt caggggggcg gagcctatgg
aaaaacgcca gcaacgcggc ctttttacgg 2280ttcctggcct tttgctggcc
ttttgctcac atgttctttc ctgcgttatc ccctgattct 2340gtggataacc
gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
2400gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
accgcctctc 2460cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
ggtttcccga ctggaaagcg 2520ggcagtgagc gcaacgcaat taatgtgagt
tagctcactc attaggcacc ccaggcttta 2580cactttatgc ttccggctcg
tatgttgtgt ggaattgtga gcggataaca atttcacaca 2640ggaaacagct
atgaccatga ttacgccaag ctttggagcc ttttttttgg agattttcaa
2700cgtgaaaaaa ttattattcg caattccttt agttgttcct ttctattctc
acagtgcaca 2760gatccaaatg acccagtctc catcctccct gtctgcatct
gtaggagaca gagtcaccat 2820cacttgccgg gcaagtcaga gcattagcag
ctacttaaat tggtatcagc agaaaccagg 2880gaaagcccct aagctcctga
tctatgctgc atccagtttg caaagtgggg tcccatcaag 2940gttcagtggc
agtggatctg ggacagattt cactctcacc atcagcagtc tgcaacctga
3000agattttgca acttactact gtcaacagag ttacagtacc cctccaacgt
tcggccaagg 3060gaccaagctc gagatcaaac gtactgtggc tgcaccatct
gtcttcatct tcccgccatc 3120tgatgagcag ttgaaatctg gaactgcctc
tgttgtgtgc ctgctgaata acttctatcc 3180cagagaggcc aaagtacagt
ggaaggtgga taacgccctc caatcgggta actcccagga 3240gagtgtcaca
gagcaggaca gcaaggacag cacctacagc ctcagcagca ccctgacgct
3300gagcaaagca gactacgaga aacacaaagt ctacgcctgc gaagtcaccc
atcagggcct 3360gagctcgccc gtcacaaaga gcttcaacag gggagagtgt
tagtaaggcg cgccaattct 3420atttcaagga gacagtcata atgaaatacc
tattgcctac ggcagccgct ggattgttat 3480tactcgcggc ccagccggcc
atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg 3540ccgcttttct
ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
3600agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
accgcttcct 3660cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
gccttctatc gccttcttga 3720cgagttcttc tgagcgggac tctggggttc
ggtgctacga gatttcgatt ccaccgccgc 3780cttctatgaa aggttgggct
tcggaatcgt tttccgggac gccggctgga tgatcctcca 3840gcgcggggat
ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa
3900tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
tttcactgca 3960ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
catgtctgta taccgtcgac 4020ctctagctag agcttggcgt aatcatggtc
atagctgttt cctgtgtgaa attgttatcc 4080gctcacaatt ccacacaaca
tacgagccgg aagcataaag tgtaaagcct ggggtgccta 4140atgagtgagc
taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
4200cctgtcgtgc cagaattgca tgaagaatct gcttagggtt aggcgttttg
cgctgcttcg 4260ctaggtggtc aatattggcc attagccata ttattcattg
gttatatagc ataaatcaat 4320attggctatt ggccattgca tacgttgtat
ccatatcata atatgtacat ttatattggc 4380tcatgtccaa cattaccgcc
atgttgacat tgattattga ctagttatta atagtaatca 4440attacggggt
cattagttca tagcccatat atggagttcc gcgttacata acttacggta
4500aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
aatgacgtat 4560gttcccatag taacgccaat agggactttc cattgacgtc
aatgggtgga gtatttacgg 4620taaactgccc acttggcagt acatcaagtg
tatcatatgc caagtacgcc ccctattgac 4680gtcaatgacg gtcaccgtct
caagcgcctc caccaagggc ccatcggtct tccccctggc 4740accctcctcc
aagagcacct ctgggggcac agcggccctg ggctgcctgg tcaaggacta
4800cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc ctgaccagcg
gcgtccacac 4860cttcccggct gtcctacagt cctcaggact ctactccctc
agcagcgtag tgaccgtgcc 4920ctccagcagc ttgggcaccc agacctacat
ctgcaacgtg aatcacaagc ccagcaacac 4980caaggtggac aagaaagttg
agcccaaatc ttgtgcggcc gcacatcatc atcaccatca 5040cggggccgca
gaacaaaaac tcatctcaga agaggatctg aatggggccg catagactgt
5100tgaaagttgt ttagcaaaac ctcatacaga aaattcattt actaacgtct
ggaaagacga 5160caaaacttta gatcgttacg ctaactatga gggctgtctg
tggaatgcta caggcgttgt 5220ggtttgtact ggtgacgaaa ctcagtgtta
cggtacatgg gttcctattg ggcttgctat 5280ccctgaaaat gagggtggtg
gctctgaggg tggcggttct gagggtggcg gttctgaggg 5340tggcggtact
aaacctcctg agtacggtga tacacctatt ccgggctata cttatatcaa
5400ccctctcgac ggcacttatc cgcctggtac tgagcaaaac cccgctaatc
ctaatccttc 5460tcttgaggag tctcagcctc ttaatacttt catgtttcag
aataataggt tccgaaatag 5520gcagggtgca ttaactgttt atacgggcac
tgttactcaa ggcactgacc ccgttaaaac 5580ttattaccag tacactcctg
tatcatcaaa agccatgtat gacgcttact ggaacggtaa 5640attcagagac
tgcgctttcc attctggctt taatgaggat ccattcgttt gtgaatatca
5700aggccaatcg tctgacctgc ctcaacctcc tgtcaatgct ggcggcggct
ctggtggtgg 5760ttctggtggc ggctctgagg gtggcggctc tgagggtggc
ggctctgagg gtggcggttc 5820tgagggtggc ggctctgagg gtggcggttc
cggtggcggc tccggttccg gtgattttga 5880ttatgaaaaa atggcaaacg
ctaataaggg ggctatgacc gaaaatgccg atgaaaacgc 5940gctacagtct
gacgctaaag gcaaacttga ttctgtcgct actgattacg gtgctgctat
6000cgatggtttc attggtgacg tttccggcct tgctaatggt aatggtgcta
ctggtgattt 6060tgctggctct aattcccaaa tggctcaagt cggtgacggt
gataattcac ctttaatgaa 6120taatttccgt caatatttac cttctttgcc
tcagtcggtt gaatgtcgcc cttatgtctt 6180tggcgctggt aaaccatatg
aattttctat tgattgtgac aaaataaact tattccgtgg 6240tgtctttgcg
tttcttttat atgttgccac ctttatgtat gtattttcga cgtttgctaa
6300catactgcgt aataaggagt cttaataaga attcactggc cgtcgtttta
caacgtcgtg 6360actgggaaaa ccctggcgtt acccaactta atcgccttgc
agcacatccc cctttcgcca 6420gctggcgtaa tagcgaagag gcccgcaccg
atcgcccttc ccaacagttg cgcagcctga 6480atggcgaatg gcgcctgatg
cggtattttc tccttacgca tctgtgcggt atttcacacc 6540gcatacgtca
aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
6600ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg
ctcctttcgc 6660tttcttccct tcctttctcg ccacgttcgc cggctttccc
cgtcaagctc taaatcgggg 6720gctcccttta gggttccgat ttagtgcttt
acggcacctc gaccccaaaa aa 67729910293DNAArtificial SequenceMV1057
99tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
60acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
120aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt
gcactctcag 180tacaatctgc tctgatgccg catagttaag ccagtatctg
ctccctgctt gtgtgttgga 240ggtcgctgag tagtgcgcga gcaaaattta
agctacaaca aggcaaggct tgaccgacaa 300ttgcatgaag aatctgctta
gggttaggcg ttttgcgctg cttcgctagg tggtcaatat 360tggccattag
ccatattatt cattggttat atagcataaa tcaatattgg ctattggcca
420ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg
tccaacatta 480ccgccatgtt gacattgatt attgactagt tattaatagt
aatcaattac ggggtcatta 540gttcatagcc catatatgga gttccgcgtt
acataactta cggtaaatgg cccgcctggc 600tgaccgccca acgacccccg
cccattgacg tcaataatga cgtatgttcc catagtaacg 660ccaataggga
ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
720gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa
tgacggtaaa 780tggcccgcct ggcattatgc ccagtacatg accttatggg
actttcctac ttggcagtac 840atctacgtat tagtcatcgc tattaccatg
gtgatgcggt tttggcagta catcaatggg 900cgtggatagc ggtttgactc
acggggattt ccaagtctcc accccattga cgtcaatggg 960agtttgtttt
ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
1020ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag
agctcgttta 1080gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt
ttgacctcca tagaagacac 1140cgggaccgat ccagcctccg cggccgggaa
cggtgcattg gaagcttggt accggtgaat 1200tggccggccc gcgccgtcga
ggttatcgat ccgaccgacg cgttcgcgag aggccgcaat 1260tccctagcca
ccatgggatg gagctgtatc atcctcttct tggtactgct gctggcccag
1320ccggccatgg ggcggagaat gggcggaact gggcggagtt aggggcggga
tgggcggagt 1380taggggcggg actatggttg ctgactaatt gagatgcgga
tccgctggca cgacaggttt 1440cccgactgga aagcgggcag tgagcgcaac
gcaattaatg tgagttagct cactcattag 1500gcaccccagg ctttacactt
tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga 1560taacaatttc
acacaggaaa cagctatgac catgattacg ccaagcttgg gctgcaggtt
1620ctttccgcct cagaagccat agagcccacc gcatccccag catgcctgct
attgtcttcc 1680caatcctccc ccttgctgtc ctgccccacc ccacccccca
gaatagaatg acacctactc 1740agacaatgcg atgcaatttc ctcattttat
taggaaagga cagtgggagt ggcaccttcc 1800agggtcaagg aaggcacggg
ggaggggcaa acaacagatg gctggcaact agaaggcaca 1860gtcgaggctg
atcagcgagc tctagatcat cgatgcatgg ggtcgtgcgc tcctttcggt
1920cgggcgctgc gggtcgtggg gcgggcgtca ggcaccgggc ttgcgggtca
tgcaccaggt 1980gcgcggtcct tcgggcacct cgacgtcggc ggtgacggtg
aagccgagcc gctcgtagaa 2040ggggaggttg cggggcgcgg aggtctccag
gaaggcgggc accccggcgc gctcggccgc 2100ctccactccg gggagcacga
cggcgctgcc cagacccttg ccctggtggt cgggcgagac 2160gccgacggtg
gccaggaacc acgcgggctc cttgggccgg tgcggcgcca ggaggccttc
2220catctgttgc tgcgcggcca gccgggaacc gctcaactcg gccatgcgcg
ggccgatctc 2280ggcgaacacc gcccccgctt cgacgctctc cggcgtggtc
cagaccgcca ccgcggcgcc 2340gtcgtccgcg acccacacct tgccgatgtc
gagcccgacg cgcgtgagga agagttcttg 2400cagctcggtc accgtctcca
gtgctagcac caagggccca tcggtcttcc ccctggcacc 2460ctcctccaag
agcacctctg ggggcacagc ggccctgggc tgcctggtca aggactactt
2520ccccgaaccg gtgacggtgt cgtggaactc aggcgccctg accagcggcg
tgcacacctt 2580cccggctgtc ctacagtcct caggactcta ctccctcagc
agcgtcgtga ccgtgccctc 2640cagcagcttg ggcacccaga cctacatctg
caacgtgaat cacaagccca gcaacaccaa 2700ggtggacaag agagttggtg
agaggccagc acagggaggg agggtgtctg ctggaagcca 2760ggctcagcgc
tcctgcctgg acgcatcccg gctatgcagt cccagtccag ggcagcaagg
2820caggccccgt ctgcctcttc acccggaggc ctctgcccgc cccactcatg
ctcagggaga 2880gggtcttctg gctttttccc caggctctgg gcaggcacag
gctaggtgcc cctaacccag 2940gccctgcaca caaaggggca ggtgctgggc
tcagacctgc caagagccat atccgggagg 3000accctgcccc tgacctaagc
ccaccccaaa ggccaaactc tccactccct cagctcggac 3060accttctctc
ctcccagatt ccagtaactc ccaatcttct ctctgcagag cccaaatctt
3120gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg
ggaccgtcag 3180tcttcctctt ccccccaaaa cccaaggaca ccctcatgat
ctcccggacc cctgaggtca 3240catgcgtggt ggtggacgtg agccacgaag
accctgaggt caagttcaac tggtacgtgg 3300acggcgtgga ggtgcataat
gccaagacaa agccgcggga ggagcagtac aacagcacgt 3360accgtgtggt
cagcgtcctc accgtcctgc accaggactg gctgaatggc aaggagtaca
3420agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc
tccaaagcca 3480aagggcagcc ccgagaacca caggtgtaca ccctgccccc
atcccgggag gagatgacca 3540agaaccaggt cagcctgacc tgcctggtca
aaggcttcta tcccagcgac atcgccgtgg 3600agtgggagag caatgggcag
ccggagaaca actacaagac cacgcctccc gtgctggact 3660ccgacggctc
cttcttcctc tatagcaagc tcaccgtgga caagagcagg tggcagcagg
3720ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac
acgcagaaga 3780gcctctccct gtctccgggt aaatgagttt aacggatctt
aattaatccg agctcggtac 3840caagcttaag tttaaaccgc tgatcagcct
cgactgtgcc ttctagttgc cagccatctg 3900ttgtttgccc ctcccccgtg
ccttccttga ccctggaagg tgccactccc actgtccttt 3960cctaataaaa
tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
4020gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
catgctgggg 4080atgcggtggg ctctatggct tctgaggcgg aaagaaccag
ctggggctct agggggtatc 4140cccacgcgcc ctgtagcggc gcattaagcg
cggcgggtgt ggtggttacg cgcagcgtga 4200ccgctacact tgccagcgcc
tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 4260cacgttcgcc
ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
4320tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
cacgtagtgg 4380gccatcgccc tgatagacgg tttttcgccc tttgacgttg
gagtccacgt tctttaatag 4440tggactcttg ttccaaactg gaacaacact
caaccctatc tcggtctatt cttttgattt 4500ataagggatt ttgccgattt
cggcctattg gttaaaaaat gagctgattt aacaaaaatt 4560taacgcgaat
taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
4620ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
gtgtggaaag 4680tccccaggct ccccagcagg cagaagtatg caaagcatgc
atctcaatta gtcagcaacc 4740atagtcccgc ccctaactcc gcccatcccg
cccctaactc cgcccagttc cgcccattct 4800ccgccccatg gctgactaat
tttttttatt tatgcagagg ccgaggccgc ctctgcctct 4860gagctattcc
agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
4920ccgggagctt ggatatccat tttcggatct gatcaagaga caggatgagg
atcgtttcgc 4980atgattgaac aagatggatt gcacgcaggt tctccggccg
cttgggtgga gaggctattc 5040ggctatgact gggcacaaca gacaatcggc
tgctctgatg ccgccgtgtt ccggctgtca 5100gcgcaggggc gcccggttct
ttttgtcaag accgacctgt ccggtgccct gaatgaactg 5160caggacgagg
cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
5220ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
gccggggcag 5280gatctcctgt catctcacct tgctcctgcc gagaaagtat
ccatcatggc tgatgcaatg 5340cggcggctgc atacgcttga tccggctacc
tgcccattcg accaccaagc gaaacatcgc 5400atcgagcgag cacgtactcg
gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 5460gagcatcagg
ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
5520ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
ggtggaaaat 5580ggccgctttt ctggattcat cgactgtggc cggctgggtg
tggcggaccg ctatcaggac 5640atagcgttgg ctacccgtga tattgctgaa
gagcttggcg gcgaatgggc tgaccgcttc 5700ctcgtgcttt acggtatcgc
cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 5760gacgagttct
tctgagcggg actctggggt tcggtgctac gagatttcga ttccaccgcc
5820gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
gatgatcctc 5880cagcgcgggg atctcatgct ggagttcttc gcccacccca
acttgtttat tgcagcttat 5940aatggttaca aataaagcaa tagcatcaca
aatttcacaa ataaagcatt tttttcactg 6000cattctagtt gtggtttgtc
caaactcatc aatgtatctt atcatgtctg tataccgtcg 6060acctctagct
agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
6120ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
ctggggtgcc 6180taatgagtga gctaactcac attaattgcg ttgcgctcac
tgcccgcttt ccagtcggga 6240aacctgtcgt gccagaattg catgaagaat
ctgcttaggg ttaggcgttt tgcgctgctt 6300cgctaggtgg tcaatattgg
ccattagcca tattattcat tggttatata gcataaatca 6360atattggcta
ttggccattg catacgttgt atccatatca taatatgtac atttatattg
6420gctcatgtcc aacattaccg ccatgttgac attgattatt gactagttat
taatagtaat 6480caattacggg gtcattagtt catagcccat atatggagtt
ccgcgttaca taacttacgg 6540taaatggccc gcctggctga ccgcccaacg
acccccgccc attgacgtca ataatgacgt 6600atgttcccat agtaacgcca
atagggactt tccattgacg tcaatgggtg gagtatttac 6660ggtaaactgc
ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
6720acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
ttatgggact 6780ttcctacttg gcagtacatc tacgtattag tcatcgctat
taccatggtg atgcggtttt 6840ggcagtacat caatgggcgt ggatagcggt
ttgactcacg gggatttcca agtctccacc 6900ccattgacgt caatgggagt
ttgttttggc accaaaatca acgggacttt ccaaaatgtc 6960gtaacaactc
cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
7020taagcagagc tcgtttagtg aaccgtcaga tcgcctggag acgccatcca
cgctgttttg 7080acctccatag aagacaccgg gaccgatcca gcctccgcgg
ccgggaacgg tgcattggaa 7140gcttggtacc ggtgaattag gcgcgccgtc
gaggttatcg atccgaccga cgcgttcgcg 7200agaggccgca attccctagc
caccatggca tgccctggct tcctgtgggc acttgtgatc 7260tccacctgtc
ttgaattctc catggctgac atccagatga cccagtctcc atcctccctg
7320tctgcatctg taggagacag agtcaccatc acttgccggg caagtcagag
cattagcagc 7380tacttaaatt ggtatcagca gaaaccaggg aaagccccta
agctcctgat ctatgctgca 7440tccagtttgc aaagtggggt cccatcaagg
ttcagtggca gtggatctgg gacagatttc 7500actctcacca tcagcagtct
gcaacctgaa gattttgcaa cttactactg tcaacagagt 7560tacagtaccc
ctccaacgtt cggccaaggg accaaggtgg agatcaaacg taagtgcact
7620ttgcggccgc taggaagaaa ctcaaaacat caagatttta aatacgcttc
ttggtctcct 7680tgctataatt atctgggata agcatgctgt tttctgtctg
tccctaacat gccctgtgat 7740tatccgcaaa caacacaccc aagggcagaa
ctttgttact taaacaccat cctgtttgct 7800tctttcctca ggaactgtgg
ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca 7860gttgaaatct
ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc
7920caaagtacag tggaaggtgg ataacgccct ccaatcgggt aactcccagg
agagtgtcac 7980agagcaggac agcaaggaca gcacctacag cctcagcagc
accctgacgc tgagcaaagc 8040agactacgag aaacacaaag tctacgcctg
cgaagtcacc catcagggcc tgagctcgcc 8100cgtcacaaag agcttcaaca
ggggagagtg ttaggtttaa cggatccgag ctcggtacca 8160agctcaagtt
taaaccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
8220gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
tgtcctttcc 8280taataaaatg aggaaattgc atcgcattgt ctgagtaggt
gtcattctat tctggggggt 8340ggggtggggc aggacagcaa gggggaggat
tgggaagaca atagcaggca tgctggggat 8400gcggtgggct ctatggcttc
tgaggcggaa agaaccagct gcattaatga atcggccaac 8460gcgcggggag
aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
8520tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
gtaatacggt 8580tatccacaga atcaggggat aacgcaggaa agaacatgtg
agcaaaaggc cagcaaaagg 8640ccaggaaccg taaaaaggcc gcgttgctgg
cgtttttcca taggctccgc ccccctgacg 8700agcatcacaa aaatcgacgc
tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 8760accaggcgtt
tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
8820ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
agctcacgct 8880gtaggtatct cagttcggtg taggtcgttc gctccaagct
gggctgtgtg cacgaacccc 8940ccgttcagcc cgaccgctgc gccttatccg
gtaactatcg tcttgagtcc aacccggtaa 9000gacacgactt atcgccactg
gcagcagcca ctggtaacag gattagcaga gcgaggtatg 9060taggcggtgc
tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
9120tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
ggtagctctt 9180gatccggcaa acaaaccacc gctggtagcg gtggtttttt
tgtttgcaag cagcagatta 9240cgcgcagaaa aaaaggatct caagaagatc
ctttgatctt ttctacgggg tctgacgctc 9300agtggaacga aaactcacgt
taagggattt tggtcatgag attatcaaaa aggatcttca 9360cctagatcct
tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
9420cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
atctgtctat 9480ttcgttcatc catagttgcc tgactccccg tcgtgtagat
aactacgata cgggagggct 9540taccatctgg ccccagtgct gcaatgatac
cgcgagaccc acgctcaccg gctccagatt 9600tatcagcaat aaaccagcca
gccggaaggg ccgagcgcag aagtggtcct gcaactttat 9660ccgcctccat
ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
9720atagtttgcg caacgttgtt
gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg 9780gtatggcttc
attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
9840tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
aagttggccg 9900cagtgttatc actcatggtt atggcagcac tgcataattc
tcttactgtc atgccatccg 9960taagatgctt ttctgtgact ggtgagtact
caaccaagtc attctgagaa tagtgtatgc 10020ggcgaccgag ttgctcttgc
ccggcgtcaa tacgggataa taccgcgcca catagcagaa 10080ctttaaaagt
gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
10140cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
tcagcatctt 10200ttactttcac cagcgtttct gggtgagcaa aaacaggaag
gcaaaatgcc gcaaaaaagg 10260gaataagggc gacacggaaa tgttgaatac tca
102931008179DNAArtificial SequencepCAGGS-IgVK1-39 targeting vector
100atccaggcgc ggatcaataa aagatcatta ttttcaatag atctgtgtgt
tggttttttg 60tgtgccttgg gggaggggga ggccagaatg aggcgcggcc aagggggagg
gggaggccag 120aatgaccttg ggggaggggg aggccagaat gaccttgggg
gagggggagg ccagaatgag 180gcgcggatcc ggagaagttc ctattccgaa
gttcctattc ttcaaatagt ataggaactt 240cgctcgaggg atcggccatt
gaacaagatg gattgcacgc aggttctccg gccgcttggg 300tggagaggct
attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
360tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
ctgtccggtg 420ccctgaatga actgcaggac gaggcagcgc ggctatcgtg
gctggccacg acgggcgttc 480cttgcgcagc tgtgctcgac gttgtcactg
aagcgggaag ggactggctg ctattgggcg 540aagtgccggg gcaggatctc
ctgtcatctc accttgctcc tgccgagaaa gtatccatca 600tggctgatgc
aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
660aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
gtcgatcagg 720atgatctgga cgaagagcat caggggctcg cgccagccga
actgttcgcc aggctcaagg 780cgcgcatgcc cgacggcgag gatctcgtcg
tgacccatgg cgatgcctgc ttgccgaata 840tcatggtgga aaatggccgc
ttttctggat tcatcgactg tggccggctg ggtgtggcgg 900accgctatca
ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
960gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
cgcatcgcct 1020tctatcgcct tcttgacgag ttcttctgag gggatcgatc
cgctgtaagt ctgcagaaat 1080tgatgatcta ttaaacaata aagatgtcca
ctaaaatgga agtttttcct gtcatacttt 1140gttaagaagg gtgagaacag
agtacctaca ttttgaatgg aaggattgga gctacggggg 1200tgggggtggg
gtgggattag ataaatgcct gctctttact gaaggctctt tactattgct
1260ttatgataat gtttcatagt tggatatcat aatttaaaca agcaaaacca
aattaagggc 1320cagctcattc ctcccactca tgatctatag atctatagat
ctctcgtggg atcattgttt 1380ttctcttgat tcccactttg tggttctaag
tactgtggtt tccaaatgtg tcagtttcat 1440agcctgaaga acgagatcag
cagcctctgt tccacataca cttcattctc agtattgttt 1500tgccaagttc
taattccatc agaagctgac tctagatggc gcgtatgcag gttttcgaca
1560ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
atagcccata 1620tatggagttc cgcgttacat aacttacggt aaatggcccg
cctggctgac cgcccaacga 1680cccccgccca ttgacgtcaa taatgacgta
tgttcccata gtaacgccaa tagggacttt 1740ccattgacgt caatgggtgg
agtatttacg gtaaactgcc cacttggcag tacatcaagt 1800gtatcatatg
ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
1860ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
acgtattagt 1920catcgctatt accatggtcg aggtgagccc cacgttctgc
ttcactctcc ccatctcccc 1980cccctcccca cccccaattt tgtatttatt
tattttttaa ttattttgtg cagcgatggg 2040ggcggggggg gggggggcgc
gcgccaggcg gggcggggcg gggcgagggg cggggcgggg 2100cgaggcggag
aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta
2160tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg
gcgggagtcg 2220ctgcgttgcc ttcgccccgt gccccgctcc gcgccgcctc
gcgccgcccg ccccggctct 2280gactgaccgc gttactccca caggtgagcg
ggcgggacgg cccttctcct ccgggctgta 2340attagcgctt ggtttaatga
cggctcgttt cttttctgtg gctgcgtgaa agccttaaag 2400ggctccggga
gggccctttg tgcggggggg agcggctcgg ggggtgcgtg cgtgtgtgtg
2460tgcgtgggga gcgccgcgtg cggcccgcgc tgcccggcgg ctgtgagcgc
tgcgggcgcg 2520gcgcggggct ttgtgcgctc cgcgtgtgcg cgaggggagc
gcggccgggg gcggtgcccc 2580gcggtgcggg ggggctgcga ggggaacaaa
ggctgcgtgc ggggtgtgtg cgtggggggg 2640tgagcagggg gtgtgggcgc
ggcggtcggg ctgtaacccc cccctgcacc cccctccccg 2700agttgctgag
cacggcccgg cttcgggtgc ggggctccgt gcggggcgtg gcgcggggct
2760cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg ggcggggcgg
ggccgcctcg 2820ggccggggag ggctcggggg aggggcgcgg cggccccgga
gcgccggcgg ctgtcgaggc 2880gcggcgagcc gcagccattg ccttttatgg
taatcgtgcg agagggcgca gggacttcct 2940ttgtcccaaa tctgtgcgga
gccgaaatct gggaggcgcc gccgcacccc ctctagcggg 3000cgcggggcga
agcggtgcgg cgccggcagg aaggaaatgg gcggggaggg ccttcgtgcg
3060tcgccgcgcc gccgtcccct tctccctctc cagcctcggg gctgtccgcg
gggggacggc 3120tgccttcggg ggggacgggg cagggcgggg ttcggcttct
ggcgtgtgac cggcggctct 3180agaagcgttg gggtgagtac tccctctcaa
aagcgggcat gacttctgcg ctaagattgt 3240cagtttccaa aaacgaggag
gatttgatat tcacctggcc cgcggtgatg cctttgaggg 3300tggccgcgtc
catctggtca gaaaagacaa tctttttgtt gtcaagcttg aggtgtggca
3360ggcttgagat ctggccatac acttgagtga cattgacatc cactttgcct
ttctctccac 3420aggtgtccac tcccagggcg gcctccggag cgatcgccga
tccgcctagg caattgttta 3480aatcggccgg ccataacttc gtataatgta
tgctatacga agttatggat cctcacagta 3540ggtggcatcg ttcctttctg
actgcccgcc ccccgcatgc cgtcccgcga tattgagctc 3600cgaacctctc
gccctgccgc cgccggtgct ccgtcgccgc cgcgccgcca tggaatcgaa
3660gccaccatgg atcttaccgg aaaactcgac gcaagaaaaa tcagagagat
cctcataaag 3720gtcaagaagg gcggaaagat cgccgtgtaa ttctagaccg
gttcgagatc caggcgcgga 3780tcaataaaag atcattattt tcaatagatc
tgtgtgttgg ttttttgtgt gccttggggg 3840agggggaggc cagaatgagg
cgcggccaag ggggaggggg aggccagaat gaccttgggg 3900gagggggagg
ccagaatgac cttgggggag ggggaggcca gaatgaggcg cgccctccgt
3960cgacctataa cttcgtataa tgtatgctat acgaagttat ggcggccgcc
accatggaca 4020tgagagtgcc cgcccagctc ctggggctcc tgctactctg
gctccgaggt aaggatggag 4080aacactagga atttactcag ccagtgtgct
cagtactgac tggaacttca gggaagttct 4140ctgataacat gattaatagt
aagaatattt gtttttatgt ttccaatctc aggtgccaga 4200tgtgacatcc
agatgaccca gagccccagc agcctgagcg ccagcgtggg cgacagagtg
4260accatcacct gcagagccag ccagagcatc agcagctacc tgaactggta
tcagcagaag 4320cccggcaagg cccccaagct gctgatctac gccgccagct
ccctgcagag cggcgtgccc 4380agcagattca gcggcagcgg ctccggcacc
gacttcaccc tgaccatcag cagcctgcag 4440cccgaggact tcgccaccta
ctactgccag cagagctaca gcaccccccc caccttcggc 4500cagggcacca
aggtggagat caagagagcc gacgccgctc ccaccgtgtc catcttcccc
4560cccagcatgg aacagctgac ctctggcgga gccaccgtgg tctgcttcgt
gaacaacttc 4620taccccagag acatcagcgt gaagtggaag atcgacggca
gcgagcagag ggacggcgtg 4680ctggacagcg tgaccgacca ggacagcaag
gactccacct acagcatgag cagcaccctg 4740agcctgacca aggtggagta
cgagaggcac aacctgtaca cctgcgaggt ggtgcacaag 4800accagctcca
gccccgtggt caagtccttc aaccggaacg agtgttgagc tagcttaaga
4860tttaaatagg ccggccgcgt cgacctcgag atccaggcgc ggatcaataa
aagatcatta 4920ttttcaatag atctgtgtgt tggttttttg tgtgccttgg
gggaggggga ggccagaatg 4980aggcgcggcc aagggggagg gggaggccag
aatgaccttg ggggaggggg aggccagaat 5040gaccttgggg gagggggagg
ccagaatgag gcgcgccccc gggtaccgag ctcgaattag 5100tggatcctca
cagtaggtgg catcgttcct ttctgactgc ccgccccccg catgccgtcc
5160cgcgatattg agctccgaac ctctcgccct gccgccgccg gtgctccgtc
gccgccgcgc 5220cgccatggaa tcgcgccggt aaccgaagtt cctatacttt
ctagagaata ggaacttcgg 5280aataggaact tcaagccggt acccagcttt
tgttcccttt agtgagggtt aatttcgagc 5340ttggcgtaat catggtcata
gctgtttcct gtgtgaaatt gttatccgct cacaattcca 5400cacaacatac
gagccgggag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
5460ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
gtcgtgccag 5520ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
tgcgtattgg gcgctcttcc 5580gcttcctcgc tcactgactc gctgcgctcg
gtcgttcggc tgcggcgagc ggtatcagct 5640cactcaaagg cggtaatacg
gttatccaca gaatcagggg ataacgcagg aaagaacatg 5700tgagcaaaag
gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
5760cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
gaggtggcga 5820aacccgacag gactataaag ataccaggcg tttccccctg
gaagctccct cgtgcgctct 5880cctgttccga ccctgccgct taccggatac
ctgtccgcct ttctcccttc gggaagcgtg 5940gcgctttctc atagctcacg
ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 6000ctgggctgtg
tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
6060cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
cactggtaac 6120aggattagca gagcgaggta tgtaggcggt gctacagagt
tcttgaagtg gtggcctaac 6180tacggctaca ctagaaggac agtatttggt
atctgcgctc tgctgaagcc agttaccttc 6240ggaaaaagag ttggtagctc
ttgatccggc aaacaaacca ccgctggtag cggtggtttt 6300tttgtttgca
agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
6360ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
tttggtcatg 6420agattatcaa aaaggatctt cacctagatc cttttaaatt
aaaaatgaag ttttaaatca 6480atctaaagta tatatgagta aacttggtct
gacagttacc aatgcttaat cagtgaggca 6540cctatctcag cgatctgtct
atttcgttca tccatagttg cctgactccc cgtcgtgtag 6600ataactacga
tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
6660ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
ggccgagcgc 6720agaagtggtc ctgcaacttt atccgcctcc atccagtcta
ttaattgttg ccgggaagct 6780agagtaagta gttcgccagt taatagtttg
cgcaacgttg ttgccattgc tacaggcatc 6840gtggtgtcac gctcgtcgtt
tggtatggct tcattcagct ccggttccca acgatcaagg 6900cgagttacat
gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
6960gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
actgcataat 7020tctcttactg tcatgccatc cgtaagatgc ttttctgtga
ctggtgagta ctcaaccaag 7080tcattctgag aatagtgtat gcggcgaccg
agttgctctt gcccggcgtc aatacgggat 7140aataccgcgc cacatagcag
aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 7200cgaaaactct
caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
7260cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
aaaaacagga 7320aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
aatgttgaat actcatactc 7380ttcctttttc aatattattg aagcatttat
cagggttatt gtctcatgag cggatacata 7440tttgaatgta tttagaaaaa
taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 7500ccacctaaat
tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
7560gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
aaagaataga 7620ccgagatagg gttgagtgtt gttccagttt ggaacaagag
tccactatta aagaacgtgg 7680actccaacgt caaagggcga aaaaccgtct
atcagggcga tggcccacta cgtgaaccat 7740caccctaatc aagttttttg
gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag 7800ggagcccccg
atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
7860agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
ctgcgcgtaa 7920ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
gtcccattcg ccattcaggc 7980tgcgcaactg ttgggaaggg cgatcggtgc
gggcctcttc gctattacgc cagctggcga 8040aagggggatg tgctgcaagg
cgattaagtt gggtaacgcc agggttttcc cagtcacgac 8100gttgtaaaac
gacggccagt gagcgcgcgt aatacgactc actatagggc gaattggggg
8160taactaagta aggatcgag 81791018188DNAArtificial
SequencepCAGGS-IgVL2-14 targeting vector 101atccaggcgc ggatcaataa
aagatcatta ttttcaatag atctgtgtgt tggttttttg 60tgtgccttgg gggaggggga
ggccagaatg aggcgcggcc aagggggagg gggaggccag 120aatgaccttg
ggggaggggg aggccagaat gaccttgggg gagggggagg ccagaatgag
180gcgcggatcc ggagaagttc ctattccgaa gttcctattc ttcaaatagt
ataggaactt 240cgctcgaggg atcggccatt gaacaagatg gattgcacgc
aggttctccg gccgcttggg 300tggagaggct attcggctat gactgggcac
aacagacaat cggctgctct gatgccgccg 360tgttccggct gtcagcgcag
gggcgcccgg ttctttttgt caagaccgac ctgtccggtg 420ccctgaatga
actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
480cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
ctattgggcg 540aagtgccggg gcaggatctc ctgtcatctc accttgctcc
tgccgagaaa gtatccatca 600tggctgatgc aatgcggcgg ctgcatacgc
ttgatccggc tacctgccca ttcgaccacc 660aagcgaaaca tcgcatcgag
cgagcacgta ctcggatgga agccggtctt gtcgatcagg 720atgatctgga
cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
780cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
ttgccgaata 840tcatggtgga aaatggccgc ttttctggat tcatcgactg
tggccggctg ggtgtggcgg 900accgctatca ggacatagcg ttggctaccc
gtgatattgc tgaagagctt ggcggcgaat 960gggctgaccg cttcctcgtg
ctttacggta tcgccgctcc cgattcgcag cgcatcgcct 1020tctatcgcct
tcttgacgag ttcttctgag gggatcgatc cgctgtaagt ctgcagaaat
1080tgatgatcta ttaaacaata aagatgtcca ctaaaatgga agtttttcct
gtcatacttt 1140gttaagaagg gtgagaacag agtacctaca ttttgaatgg
aaggattgga gctacggggg 1200tgggggtggg gtgggattag ataaatgcct
gctctttact gaaggctctt tactattgct 1260ttatgataat gtttcatagt
tggatatcat aatttaaaca agcaaaacca aattaagggc 1320cagctcattc
ctcccactca tgatctatag atctatagat ctctcgtggg atcattgttt
1380ttctcttgat tcccactttg tggttctaag tactgtggtt tccaaatgtg
tcagtttcat 1440agcctgaaga acgagatcag cagcctctgt tccacataca
cttcattctc agtattgttt 1500tgccaagttc taattccatc agaagctgac
tctagatggc gcgtatgcag gttttcgaca 1560ttgattattg actagttatt
aatagtaatc aattacgggg tcattagttc atagcccata 1620tatggagttc
cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
1680cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
tagggacttt 1740ccattgacgt caatgggtgg agtatttacg gtaaactgcc
cacttggcag tacatcaagt 1800gtatcatatg ccaagtacgc cccctattga
cgtcaatgac ggtaaatggc ccgcctggca 1860ttatgcccag tacatgacct
tatgggactt tcctacttgg cagtacatct acgtattagt 1920catcgctatt
accatggtcg aggtgagccc cacgttctgc ttcactctcc ccatctcccc
1980cccctcccca cccccaattt tgtatttatt tattttttaa ttattttgtg
cagcgatggg 2040ggcggggggg gggggggcgc gcgccaggcg gggcggggcg
gggcgagggg cggggcgggg 2100cgaggcggag aggtgcggcg gcagccaatc
agagcggcgc gctccgaaag tttcctttta 2160tggcgaggcg gcggcggcgg
cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg 2220ctgcgttgcc
ttcgccccgt gccccgctcc gcgccgcctc gcgccgcccg ccccggctct
2280gactgaccgc gttactccca caggtgagcg ggcgggacgg cccttctcct
ccgggctgta 2340attagcgctt ggtttaatga cggctcgttt cttttctgtg
gctgcgtgaa agccttaaag 2400ggctccggga gggccctttg tgcggggggg
agcggctcgg ggggtgcgtg cgtgtgtgtg 2460tgcgtgggga gcgccgcgtg
cggcccgcgc tgcccggcgg ctgtgagcgc tgcgggcgcg 2520gcgcggggct
ttgtgcgctc cgcgtgtgcg cgaggggagc gcggccgggg gcggtgcccc
2580gcggtgcggg ggggctgcga ggggaacaaa ggctgcgtgc ggggtgtgtg
cgtggggggg 2640tgagcagggg gtgtgggcgc ggcggtcggg ctgtaacccc
cccctgcacc cccctccccg 2700agttgctgag cacggcccgg cttcgggtgc
ggggctccgt gcggggcgtg gcgcggggct 2760cgccgtgccg ggcggggggt
ggcggcaggt gggggtgccg ggcggggcgg ggccgcctcg 2820ggccggggag
ggctcggggg aggggcgcgg cggccccgga gcgccggcgg ctgtcgaggc
2880gcggcgagcc gcagccattg ccttttatgg taatcgtgcg agagggcgca
gggacttcct 2940ttgtcccaaa tctgtgcgga gccgaaatct gggaggcgcc
gccgcacccc ctctagcggg 3000cgcggggcga agcggtgcgg cgccggcagg
aaggaaatgg gcggggaggg ccttcgtgcg 3060tcgccgcgcc gccgtcccct
tctccctctc cagcctcggg gctgtccgcg gggggacggc 3120tgccttcggg
ggggacgggg cagggcgggg ttcggcttct ggcgtgtgac cggcggctct
3180agaagcgttg gggtgagtac tccctctcaa aagcgggcat gacttctgcg
ctaagattgt 3240cagtttccaa aaacgaggag gatttgatat tcacctggcc
cgcggtgatg cctttgaggg 3300tggccgcgtc catctggtca gaaaagacaa
tctttttgtt gtcaagcttg aggtgtggca 3360ggcttgagat ctggccatac
acttgagtga cattgacatc cactttgcct ttctctccac 3420aggtgtccac
tcccagggcg gcctccggag cgatcgccga tccgcctagg caattgttta
3480aatcggccgg ccataacttc gtataatgta tgctatacga agttatggat
cctcacagta 3540ggtggcatcg ttcctttctg actgcccgcc ccccgcatgc
cgtcccgcga tattgagctc 3600cgaacctctc gccctgccgc cgccggtgct
ccgtcgccgc cgcgccgcca tggaatcgaa 3660gccaccatgg atcttaccgg
aaaactcgac gcaagaaaaa tcagagagat cctcataaag 3720gtcaagaagg
gcggaaagat cgccgtgtaa ttctagaccg gttcgagatc caggcgcgga
3780tcaataaaag atcattattt tcaatagatc tgtgtgttgg ttttttgtgt
gccttggggg 3840agggggaggc cagaatgagg cgcggccaag ggggaggggg
aggccagaat gaccttgggg 3900gagggggagg ccagaatgac cttgggggag
ggggaggcca gaatgaggcg cgccctccgt 3960cgacctataa cttcgtataa
tgtatgctat acgaagttat ggcggccgcc accatggaca 4020tgagagtgcc
cgcccagctc ctggggctcc tgctactctg gctccgaggt aaggatggag
4080aacactagga atttactcag ccagtgtgct cagtactgac tggaacttca
gggaagttct 4140ctgataacat gattaatagt aagaatattt gtttttatgt
ttccaatctc aggtgccaga 4200tgtcagtctg ccctgaccca gcccgcctct
gtgtctggca gccctggcca gagcatcacc 4260atcagctgca ccggcaccag
cagcgacgtg ggcggctaca actacgtgtc ctggtatcag 4320cagcaccccg
gcaaggcccc caagctgatg atctacgagg tgtccaacag acccagcggc
4380gtgagcaaca gattcagcgg cagcaagagc ggcaacaccg ccagcctgac
catcagcggc 4440ctccaggctg aggacgaggc cgactactac tgcagcagct
acaccagcag ctccaccctg 4500gtgtttggcg gcggaacaaa gctgaccgtg
ctgagagccg acgccgctcc caccgtgtcc 4560atcttccccc ccagcatgga
acagctgacc tctggcggag ccaccgtggt ctgcttcgtg 4620aacaacttct
accccagaga catcagcgtg aagtggaaga tcgacggcag cgagcagagg
4680gacggcgtgc tggacagcgt gaccgaccag gacagcaagg actccaccta
cagcatgagc 4740agcaccctga gcctgaccaa ggtggagtac gagaggcaca
acctgtacac ctgcgaggtg 4800gtgcacaaga ccagctccag ccccgtggtc
aagtccttca accggaacga gtgttgagct 4860agcttaagat ttaaataggc
cggccgcgtc gacctcgaga tccaggcgcg gatcaataaa 4920agatcattat
tttcaataga tctgtgtgtt ggttttttgt gtgccttggg ggagggggag
4980gccagaatga ggcgcggcca agggggaggg ggaggccaga atgaccttgg
gggaggggga 5040ggccagaatg accttggggg agggggaggc cagaatgagg
cgcgcccccg ggtaccgagc 5100tcgaattagt ggatcctcac agtaggtggc
atcgttcctt tctgactgcc cgccccccgc 5160atgccgtccc gcgatattga
gctccgaacc tctcgccctg ccgccgccgg tgctccgtcg 5220ccgccgcgcc
gccatggaat cgcgccggta accgaagttc ctatactttc tagagaatag
5280gaacttcgga ataggaactt caagccggta cccagctttt gttcccttta
gtgagggtta 5340atttcgagct tggcgtaatc atggtcatag ctgtttcctg
tgtgaaattg ttatccgctc 5400acaattccac acaacatacg agccgggagc
ataaagtgta aagcctgggg tgcctaatga 5460gtgagctaac tcacattaat
tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 5520tcgtgccagc
tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
5580cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
gcggcgagcg 5640gtatcagctc actcaaaggc ggtaatacgg ttatccacag
aatcagggga taacgcagga 5700aagaacatgt gagcaaaagg ccagcaaaag
gccaggaacc gtaaaaaggc cgcgttgctg 5760gcgtttttcc ataggctccg
cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 5820aggtggcgaa
acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
5880gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
tctcccttcg 5940ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
tcagttcggt gtaggtcgtt 6000cgctccaagc tgggctgtgt gcacgaaccc
cccgttcagc ccgaccgctg cgccttatcc 6060ggtaactatc gtcttgagtc
caacccggta agacacgact tatcgccact ggcagcagcc 6120actggtaaca
ggattagcag
agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 6180tggcctaact
acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca
6240gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
cgctggtagc 6300ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
aaaaaggatc tcaagaagat 6360cctttgatct tttctacggg gtctgacgct
cagtggaacg aaaactcacg ttaagggatt 6420ttggtcatga gattatcaaa
aaggatcttc acctagatcc ttttaaatta aaaatgaagt 6480tttaaatcaa
tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
6540agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
ctgactcccc 6600gtcgtgtaga taactacgat acgggagggc ttaccatctg
gccccagtgc tgcaatgata 6660ccgcgagacc cacgctcacc ggctccagat
ttatcagcaa taaaccagcc agccggaagg 6720gccgagcgca gaagtggtcc
tgcaacttta tccgcctcca tccagtctat taattgttgc 6780cgggaagcta
gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
6840acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
cggttcccaa 6900cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
aagcggttag ctccttcggt 6960cctccgatcg ttgtcagaag taagttggcc
gcagtgttat cactcatggt tatggcagca 7020ctgcataatt ctcttactgt
catgccatcc gtaagatgct tttctgtgac tggtgagtac 7080tcaaccaagt
cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
7140atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
tggaaaacgt 7200tcttcggggc gaaaactctc aaggatctta ccgctgttga
gatccagttc gatgtaaccc 7260actcgtgcac ccaactgatc ttcagcatct
tttactttca ccagcgtttc tgggtgagca 7320aaaacaggaa ggcaaaatgc
cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 7380ctcatactct
tcctttttca atattattga agcatttatc agggttattg tctcatgagc
7440ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
cacatttccc 7500cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt
aaaattcgcg ttaaattttt 7560gttaaatcag ctcatttttt aaccaatagg
ccgaaatcgg caaaatccct tataaatcaa 7620aagaatagac cgagataggg
ttgagtgttg ttccagtttg gaacaagagt ccactattaa 7680agaacgtgga
ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
7740gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
ctaaatcgga 7800accctaaagg gagcccccga tttagagctt gacggggaaa
gccggcgaac gtggcgagaa 7860aggaagggaa gaaagcgaaa ggagcgggcg
ctagggcgct ggcaagtgta gcggtcacgc 7920tgcgcgtaac caccacaccc
gccgcgctta atgcgccgct acagggcgcg tcccattcgc 7980cattcaggct
gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
8040agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca
gggttttccc 8100agtcacgacg ttgtaaaacg acggccagtg agcgcgcgta
atacgactca ctatagggcg 8160aattgggggt aactaagtaa ggatcgag
818810210DNAArtificial SequenceKozak sequence 102gccaccatgg 10
* * * * *